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A      TEXTBOOK      OF 

BACTERIOLOGY 


A    TEXTBOOK    OF 

B ACT  E  R I OLOG Y 

A  PRACTICAL  TREATISE  FOR   STUDENTS 

AND  PRACTITIONERS  OF  MEDICINE 

AND    PUBLIC    HEALTH 


BY 

HANS   ZINSSER,   M.D. 

PROFESSOR  OF  BACTERIOLOGY,    COLLEGE   OF  PHYSICIANS  AND   SURGEONS,   COLUMBIA    UNI- 
VERSITY, NEW  YORK  CITY;  BACTERIOLOGIST  TO  THE  PRESBYTERIAN  HOSPITAL; 
FORMERLY  PROFESSOR  OF  BACTERIOLOGY  AND  IMMUNITY,    STANFORD 
UNIVERSITY,  CALIFORNIA;  COLONEL  MEDICAL  OFFICERS'  '    ' 

RESERVE  CORPS,  U.  S.  A.  •    •  Y  »     *( 


WITH  A  SECTION  ON  PATHOGENIC  PROTOZOA 
BY 

FREDERICK  P.  RUSSELL,  M.D. 

BRIGADIER  GENERAL,    MEDICAL  OFFICERS'  RESERVE  CORPS,   U.   8.    A.,  FORMERLY 

PROFESSOR  OF  BACTERIOLOGY   AND  PATHOLOGY,    ARMY   MEDICAL 

SCHOOL  AND  GEORGE  WASHINGTON  UNIVERSITY 

(Completely  revised  and  rewritten  from  the  original  text 
of  Hiss  and  Zinsser) 

WITH  ONE  HUNDRED  AND  SIXTY-FOUR  ILLUSTRATIONS 
IN  THE  TEXT 

FIFTH   EDITION 


D.   APPLETON   AND   COMPANY 

NEW  YORK       ::       1922       :;       LONDON 


COPYIUGHT,  1910,  191-1,  1916,  1918,  and  1922,  BY 

D.  APPLETON  AND  COMPANY 


our 


ITY    T<1-' 


PRINTED   IN   THE    UNITED   STATES  OF   AMERICA 


PREFACE  TO  THE  FIRST  EDITION 

THE  volume  here  presented  is  primarily  a  treatise  on  the  funda- 
mental laws  and  technique  of  Bacteriology,  as  illustrated  by  their 
application  to  the  study  of  pathogenic  bacteria. 

So  ubiquitous  are  the  bacteria  and  so  manifold  their  activities 
that  Bacteriology,  although  one  of  the  youngest  of  sciences,  has 
already  been  divided  into  special  fields — Medical,  Sanitary,  Agricul- 
tural, and  Industrial — having  little  in  common,  except  problems  of 
general  bacterial  physiology  and  certain  fundamental  technical 
procedures. 

From  no  other  point  of  approach,  however,  is  such  a  breadth  of 
conception  attainable,  as  through  the  study  of  bacteria  in  their 
relation  to  disease  processes  in  man  and  animals.  Through  such  a 
study  one  must  become  familiar  not  only  with  the  growth  character- 
istics and  products  of  the  bacteria  apart  from  the  animal  body,  thus 
gaining  a  knowledge  of  methods  and  procedures  common  to  the 
study  of  pathogenic  and  non-pathogenic  organisms,  but  also  with 
those  complicated  reactions  taking  place  between  the  bacteria  and 
their  products  on  the  one  hand  and  the  cells  and  fluids  of  the  animal 
body  on  the  other — reactions  which  often  manifest  themselves  as 
symptoms  and  lesions  of  disease  or  by  visible  changes  in  the  test  tube. 

Through  a  study  and  comprehension  of  the  processes  underlying 
these  reactions,  our  knowledge  of  cell  physiology  has  been  broadened, 
and  facts  of  inestimable  value  have  been  discovered,  which  have 
thrown  light  upon  some  of  the  most  obscure  problems  of  infection 
and  immunity  and  have  led  to  hitherto  unsuspected  methods  of 
treatment  and  diagnosis.  Thus,  through  Medical  Bacteriology — that 
highly  specialized  offshoot  of  General  Biology  and  Pathology — have 
been  given  back  to  the  parent  sciences  and  to  Medicine  in  general 
methods  and  knowledge  of  the  widest  application. 

It  has  been  our  endeavor,  therefore,  to  present  this  phase  of  our 
subject  in  as  broad  and  critical  a  manner  as  possible  in  the  sections 
dealing  with  infection  and  immunity  and  with  methods  of  biological 
diagnosis  and  treatment  of  disease,  so  that  the  student  and  practi- 

47T0203 


vi  PREFACE  TO  THE  FIRST  EDITION 

tioner  of  medicine,  by  becoming  familiar  with  underlying  laws  and 
principles,  may  not  only  be  in  a  position  to  realize  the  meaning  and 
scope  of  some  of  these  newer  discoveries  and  methods,  but  may  be 
in  better  position  to  decide  for  themselves  their  proper  application 
and  limitations. 

We  have  not  hesitated,  whenever  necessary  for  a  proper  under- 
standing of  processes  of  bacterial  nutrition  or  physiology,  or  for 
breadth  of  view  in  considering  problems  of  the  relation  of  bacteria 
to  our  food  supply  and  environment,  to  make  free  use  of  illustrations 
from  the  more  special  fields  of  agricultural  and  sanitary  bacteriology, 
and  some  special  methods  of  the  bacteriology  of  sanitation  are  given 
in  the  last  division  of  the  book,  dealing  with  the  bacteria  in  relation 
to  our  food  and  environment. 

In  conclusion  it  may  be  said  that  the  scope  and  arrangement  of 
subjects  treated  of  in  this  book  are  the  direct  outcome  of  many  years 
of  experience  in  the  instruction  of  students  in  medical  and  in 
advanced  university  courses  in  bacteriology,  and  that  it  is  our  hope 
that  this  volume  may  not  only  meet  the  needs  of  such  students 
but  may  prove  of  value  to  the  practitioner  of  medicine  for  whom 
it  has  also  been  written. 

It  is  a  pleasure  to  acknowledge  the  courtesy  of  those  who 
furnished  us  with  illustrations  for  use  in  the  text,  and  our  indebted- 
ness to  Dr.  Gardner  Hopkins  and  Professor  Francis  Carter  Wood 
for  a  number  of  the  photomicrographs  taken  especially  for  this  work. 

P.  H.  H.,  JR., 
H.  Z. 


PREFACE  TO  THE  FIFTH  EDITION 

THE  present  or  Fifth  Edition  of  the  TEXTBOOK  OF  BACTERIOLOGY 
represents  an  almost  complete  rewriting  of  the  book.  The  four 
preceding  editions  were  in  each  case  altered  and  brought  up  to 
date  from  time  to  time,  but  in  all  of  them  the  original  plan  of  presen- 
tation, conceived  at  the  first  writing,  was  preserved.  In  the  present 
edition  many  and  important  additions  of  material  and  changes  in 
manner  of  presentation  have  been  made. 

Bacteriology  and  the  reasoning  based  on  bacteriological  and  im- 
munological  discoveries  have  become  more  and  more  closely  interwoven 
with  the  clinical  and  public  health  aspects  of  infectious  diseases. 
Indeed,  if  this  had  not  been  sufficiently  apparent  before  1914,  the 
experiences  of  the  late  war  have  demonstrated,  conclusively,  how  im- 
possible it  is  to  organize  either  hospitals  for  infectious  diseases  or 
organizations  for  the  control  of  epidemics  without  the  intimate 
participation  of  men  trained  in  bacteriology.  It  seems  to  us,  also, 
to  have  become  apparent  that  the  bacteriologist  who  takes  an  active 
part  in  the  work  of  a  hospital  or  in  directing  sanitary  undertakings, 
must  have  a  very  thorough  understanding  of  the  clinical  and  public 
health  aspects  of  the  problem  as  a  whole. 

The  conception  upon  which  the  preparation  of  the  new  edition  of 
the  book  has  been  based,  therefore,  is  the  belief  that  no  thorough 
understanding  of  the  clinical  problems  of  infectious  disease  or  of 
larger  public  health  measures  can  be  attained  without  thorough 
familiarity  with  the  bacteriological  and  immunological  facts  upon 
which  clinical  and  sanitary  reasoning  must  be  based.  The  book 
represents,  therefore,  in  a  brief  way,  an  attempt  to  correlate  a  labora- 
tory knowledge  with  the  branches  of  medicine  and  prophylaxis  to 
which  it  is  most  directly  applicable. 

We  have  felt  that  a  Textbook  of  Bacteriology,  primarily  aimed  at 
the  needs  of  medical  students  and  physicians,  may  be  regarded 
as  neglecting  a  great  opportunity  or,  perhaps,  even  an  obligation,  if 
it  omits  emphasis  upon  prevention.  In  order  to  accomplish  this  purpose 
it  has  been  necessary  to  add  brief  clinical  data  in  the  case  of  each 

vii 


vin  PREFACE   TO  THE  FIFTH  EDITION 

variety  of  infection  and  to  incorporate  in  the  more  important  chapters, 
brief  discussions  of  the  principles  underlying  sanitary  procedure. 

In  the  sections  on  technique  we  have  eliminated  many  methods 
which  we  have  ceased  to  use  ourselves.  At  the  laboratory  of  the 
Bacteriological  Department  of  the  Medical  School  of  Columbia  Uni- 
versity we  have  had  the  opportunity  of  having  advanced  students 
and  staff  try  out  a  great  many  bacteriological  procedures  and  have, 
in  consequence,  been  able  to  eliminate  a  number  of  methods  that,  as 
matters  of  routine,  have  been  practically  dropped  from  our  practice. 
We  have  considerably  simplified  the  section  on  media.  The  newer 
methods  of  titration  have  been  added. 

The  immunological  section  has  been  considerably  changed  and  we 
think  simplified.  It  is  not  the  purpose  of  a  book  of  this  kind  to  pre- 
sent a  critical  thesis  on  theoretical  immunity.  We  have,  therefore,  re- 
stricted ourselves  to  the  exposition  of  the  more  important  principles 
and  practical  methods  needed  by  routine  laboratory  workers.  Short 
sections  on  the  normal  bacterial  flora  of  the  human  body  have  been, 
added,  with  particular  consideration  of  the  important  work  done  in 
recent  years  by  Herter,  Kendall,  Rettger  and  others.  Most  of  the 
sections  dealing  with  the  pathogenic  microorganisms  themselves  have 
been  completely  rewritten  and  the  order  of  presentation  consid- 
erably altered  in  order  to  bring  together,  more  logically,  infections 
which  are  usually  considered  together  from  the  clinical  point  of 
view.  The  diagnostic  and  therapeutic  principles  in  which  bacteri- 
ological and  immunological  reasoning  and  technique  are  involved  have 
been  thoroughly  dealt  with. 

The  section  on  Protozoa  has  been  completely  revised  by  one  of  us 
along  the  same  general  lines  adopted  for  the  revision  of  the  bacteri- 
ological section. 

The  writers  realize  that  the  inclusion  of  clinical  and  epidemio- 
logical  data  in  a  Textbook  of  Bacteriology  is  considerably  at  variance 
with  the  usual  treatment  given  to  the  subject  in  books  of  this  kind. 
But  it  is  hoped  that  this  manner  of  treatment  will  add  considerably  to 
the  value  of  the  book  for  those  who  are  interested  in  microorganisms 
particularly  in  their  relationship  to  clinical  and  preventive  medicine. 

In  conclusion  great ful  acknowledgment  is  made  to  a  number  of 
our  associates  in  the  Department  of  Bacteriology  at  Columbia  for 
valuable  aid  in  the  preparation  of  this  Edition.  Dr.  J.  0.  Hopkins, 
Associate  in  the  Department  who  lias  boon  working  with  the  patho- 
genic molds,  has  rewritten  the  section  dealing  with  these  organisms. 


PREFACE   TO   THE   FIFTH   EDITION  ix 

Dr.  J.  Howard  Mueller,  Assistant  Professor  of  Bacteriology,  has 
critically  revised  the  sections  dealing  with  the  chemical  metabolism  of 
bacteria,  and  Miss  Ann  KuttniT,  Instructor  in  the  department,  has 
elaborated,  and  revised  the  chapter  dealing  with  the  anaerobic-  in- 
fections which  have  gained  such  an  important  place  in  the  study  of 
traumatic  injuries.  We  are  indebted  to  Dr.  Oscar  Tcague  for  valuable 
suggestions  in  connection  with  the  chapters  on  cholera  and  plague. 

HANS  ZINSSER 
FREDERICK  F.  RUSSELL 


CONTENTS 

SECTION  I 

THE  GENERAL  BIOLOGY  OF  BACTERIA  AND  THE 
TECHNIQUE  OF  BACTERIOLOGICAL  STUDY 

CHAPTER  PAGE 

I.  THE  DEVELOPMENT  AND  SCOPE  OF  BACTERIOLOGY 1 

II.  GENERAL  MORPHOLOGY,  REPRODUCTION,  AND  CHEMICAL  AND  PHYSICAL 

PROPERTIES  OF  THE  BACTERIA 9 

III.  THE  RELATION  OF  BACTERIA  TO  ENVIRONMENT,  AND  THEIR  CLASSI- 

FICATION   27 

IV.  THE  BIOLOGICAL  ACTIVITIES  OF  BACTERIA 45 

V.  'THE  DESTRUCTION  OF  BACTERIA 76 

VI.  METHODS  USED  IN  THE  MICROSCOPIC  STUDY  AND  STAINING  OF  BAC- 
TERIA   Ill 

VII.  THE  PREPARATION  OF  CULTURE  MEDIA 133 

VIII.  METHODS  USED  IN  THE  CULTIVATION  OF  BACTERIA    .      .      .      .      .      .    172 

IX.  METHODS  DETERMINING  BIOLOGICAL  ACTIVITIES  OF  BACTERIA,  ANIMAL 

EXPERIMENTATION 196 

X.  THE  BACTERIOLOGICAL  EXAMINATION  OF  MATERIAL  FROM  PATIENTS 

AND  OUTLINE  OF  FLORA  OF  THE  NORMAL  HUMAN  BODY   .  .   206 


SECTION  II 
INFECTION  AND  IMMUNITY 

CHAPTER  PAGE 

XI.  FUNDAMENTAL  FACTORS  OF  PATHOGENICITY  AND  INFECTION     .      .      .  230 

XII.  DEFENSIVE  FACTORS  OF  THE  ANIMAL  ORGANISM 240 

XIII.  TOXINS  AND  ANTITOXINS 255 

XIV.  PRODUCTION  AND  TESTING  OF  ANTITOXINS 269 

XV.  SENSITIZING    ANTIBODIES    (PHENOMENA    OF    LYSIS,    AGGLUTINATION, 

PRECIPITATION,  ETC.) 277 

XVI.  THE  TECHNIQUE  OF  SERUM  REACTIONS 301 

XVII.  PHAGOCYTOSIS  .  .   330 


xiv  CONTENTS 


SECTION  VI 
BACTERIA  IN  AIR,  SOIL,  WATER  AND  MILK 

CHAPTER  PAGE 

LI.  BACTERIA  IN  THE  AIR  AND  SOIL 1010 

III.  BACTERIA  IN  WATER  1016 

LIII.  BACTERIA   IN    MILK   AND    MILK   PRODUCTS.     BACTERIA    IN   THE 

INDUSTRIES  .  1027 


SECTION  VII 
PATHOGENIC  PROTOZOA 

CHAPTER  PAGE 

LIV.  THE  AMCEB^J 1050 

LV.  MASTIGOPHORA        1073 

LVI.  SPOROZOA 1098 

LVII.  INFUSORIA          1137 

LVIII.  TECHNIQUE  OF  BLOOD  EXAMINATIONS  FOR  PROTOZOA       ....  1140 

INDEX  OF  AUTHORS 1145 

INDEX  OF  SUBJECTS  .  .1159 


SECTION   I 

THE    GENERAL    BIOLOGY    OF    BACTERIA    AND    THE 
TECHNIQUE    OF    BACTERIOLOGICAL   STUDY 


CHAPTER  I 
THE  DEVELOPMENT  AND  SCOPE  OF  BACTERIOLOGY 

As  we  trace  back  to  their  ultimate  origins  the  lines  of  develop- 
ment of  living  beings  of  the  animal  and  plant  kingdoms,  we  find  them 
converging  toward  a  common  type,  represented  by  a  large  group  of 
unicellular  organisms,  so  simple  in  structure,  so  unspecialized  in 
function,  that  their  classification  in  either  the  realm  of  plants  or  that 
of  animals  becomes  little  more  than  an  academic  question.  How- 
ever, even  such  microorganisms,  in  which  the  functions  of  nutrition, 
respiration,  locomotion,  and  reproduction  are  concentrated  within 
the  confines  of  a  single  cell,  and  in  which  adaptation  to  special  con- 
ditions more  readily  brings  about  modifications  leading  to  the  pro- 
duction of  a  multitude  of  delicately  graded  transitional  forms,  fall 
into  groups  which,  either  in  structure  or  in  biological  attributes  show 
evidence  of  a  tendency  toward  one  or  the  other  of  the  great  kingdoms. 

Most  important  of  these  unicellular  forms,  for  the  student  of 
medical  science,  are  the  bacteria  and  the  protozoa. 

The  former,  by  reason  of  their  undifferentiated  protoplasm,  their 
occasional  possession  of  cellulose  membranes,  their  biological  ten- 
dency to  synthetize,  as  well  as  to  break  down  organic  compounds, 
and  because  of  the  transitional  forms  which  seem  to  connect  them 
directly  with  the  lower  plants,  are  generally  placed  in  the  plant 
kingdom.  The  latter,  chiefly  on  the  basis  of  metabolism,  are  classi- 
fied with  the  animals. 

Knowledge  of  the  existence  of  microorganisms  as  minute  as  the 
ones  under  discussion  was  of  necessity  forced  to  await  the  perfec- 
tion of  instruments  of  magnification.  It  was  not  until  the  latter  half 
of  the  seventeenth  century,  therefore,  that  the  Jesuit,  Kircher,  in 


.*>    BIOLOGY  AND  TECHNIQUE 


1659,  and  the  Dutch  linen-draper,  van  Leeuwenhoek,  in  1875,  actually 
saw  and  described  living  beings  too  small  to  be  seen  with  the  naked 
eye.  There  can  be  no  doubt  that  the  small  bodies  seen  by  these  men 
and  their  many  immediate  successors  were,  at  least  in  part,  bacteria. 
And  indeed  the  descriptions  and  illustrations  of  several  of  the 
earliest  workers  correspond  with  many  of  the  forms  which  are  well 
known  to  us  at  the  present  day. 

During  the  century  following  the  work  of  these  pioneers,  the 
efforts  of  investigators  lay  chiefly  in  the  more  exact  morphological 
description  of  some  of  the  forms  of  unicellular  life,  already  known. 
Conspicuous  among  the  work  of  this  period  is  that  of  Otto  Friedrich 
Muller.  In  the  generation  following  Miiller's  work,  however,  a 
marked  advance  in  the  study  of  these  forms  was  made  by  Ehren- 
berg,1  who  established  a  classification  which,  in  some  of  its  cardinal 
divisions,  is  retained  until  the  present  day. 

Meanwhile  the  regularity  with  which  these  ' '  animalcula "  or 
"infusion  animalcula"  were  demonstrable  in  tartar  from  the  teeth, 
in  intestinal  contents,  in  well-water,  etc.,  had  begun  to  arouse  in  the 
minds  of  the  more  advanced  physicians  of  the  time  a  suspicion  as  to 
a  possible  relationship  of  these  minute  forms  with  disease.  The  con- 
ception of  * '  contagion, ' '  or  transmission  of  a  disease  from  one  human 
being  to  another,  was,  however,  even  at  this  time,  centuries  old.  The 
fact  had  been  recognized  by  Aristotle,  had  been  reiterated  by 
medieval  philosophers,  and  had  led,  in  1546,  to  the  division  of  con- 
tagious diseases  by  Fracastor,  into  those  transmitted  "per  contac- 
tum,"  and  those  conveyed  indirectly  "per  fomitem."  It  was  for 
these  mysterious  facts  of  the  transmissibility  of  disease,  that  clini- 
cians of  the  eighteenth  century,  with  remarkable  insight,  saw  an 
explanation  in  the  microorganisms  discovered  by  Leeuwenhoek  and 
his  followers. 

In  fact,  Plenciz  of  Vienna,  writing  in  1762,  not  only  expressed  a 
belief  in  the  direct  etiological  connection  between  microorganisms 
and  some  diseases,  but  was  the  first  to  advance  the  opinion  that  each 
malady  had  its  own  specific  causal  agent,  which  multiplied  enor- 
mously in  the  diseased  body.  The  opinions  of  this  author,  if  trans- 
lated into  the  language  of  our  modern  knowledge  of  the  subject, 
came  remarkably  near  to  the  truth,  not  only  as  regards  etiology  and 
transmission,  but  also  in  their  suggestion  of  specific  therapy. 

The  conception  of  a  "contagium  vivum"  was  thus  practically 

1"Die  Infusionstierchen, "  etc.,  Leipzig,  1838. 


DEVELOPMENT  AND  SCOPE  OF  BACTERIOLOGY  3 

established  with  the  work  of  Plenciz  and  many  others  who  followed 
in  his  train,  but  the  astonishingly  slight  impression  which  the  acute 
reasoning  of  these  men  left  upon  the  medical  thought  of  their  day 
is  illustrative  of  the  futility  of  the  most  penetrating  speculation 
when  unsupported  by  experimental  data. 

The  real  advancement  in  the  scientific  development  of  the  subject 
was  achieved  along  entirely  different  lines.  In  1837,  Schwann,  a 
botanist,  showed  that  the  yeasts,  found  in  fermenting  substances, 
were  living  beings,  which  bore  a  causal  relationship  to  the  process 
of  fermentation.  At  almost  the  same  time,  similar  observations  were 
made  by  a  French  physicist,  Cagniard-Latour.  The  opinions  ad- 
vanced by  these  men  on  the  nature  of  fermentation  aroused  much 
interest  and  discussion,  since,  at  that  time  and  for  a  long  period 
thereafter,  fermentation  was  ascribed  universally  to  protein  decom- 
position, a  process  which  was  entirely  obscure  but  firmly  believed  to 
be  of  a  purely  chemical  nature. 

Although  belief  in  the  discovery  of  Schwann  did  not  master  the 
field  until  after  Pasteur  had  completed  his  classical  studies  upon  the 
fermentations  occurring  in  beer  and  wine,  yet  the  conception  of  a 
"fermentum  vivum"  aroused  much  speculation,  and  the  attention  of 
physicians  and  scientists  was  attracted  to  the  many  analogies  exist- 
ing between  phenomena  of  fermentation  and  those  of  disease. 

The  conception  of  such  an  analogy,  however,  was  not  a  new 
thought  in  the  philosophy  of  the  time.  Long  before  Schwann  and 
Cagniard-Latour,  the  philosopher  Robert  Boyle,  working  in  the 
seventeenth  century,  had  prophesied  that  the  mystery  of  infectious 
diseases  would  be  solved  by  him  who  should  succeed  in  elucidating 
the  nature  of  fermentation. 

Nevertheless,  the  diligent  search  for  microorganisms  in  relation 
to  various  diseases  which  followed  led  to  few  results,  and  the  suc- 
cesses which  were  attained  were  limited  to  the  diseases  caused  by 
some  of  the  larger  fungi,  favus  (1839),  thrush  (1839),  and  pityriasis 
versicolor  (1846).  During  this  time  of  ardent  but  often  poorly  con- 
trolled etiological  research,  it  was  Henle  who  formulated  the  postu- 
lates of  conservatism,  almost  as  rigid  as  the  later  postulates  of  Koch, 
requiring  that  proof  of  the  etiological  relationship  of  a  microorgan- 
ism to  a  disease  could  not  be  brought  merely  by  finding  it  in  a  lesion 
of  the  disease,  but  that  constant  presence  in  such  lesions  must  be 
proven  and  isolation  and  study  of  the  microorganism  away  from  the 
diseased  body  must  be  carried  out. 


4  BIOLOGY  AND  TECHNIQUE 

It  was  during  this  period  also  that  one  of  the  most  fundamental 
questions,  namely,  that  of  the  origin  of  these  minute  living  beings, 
was  being  discussed  with  much  passion  by  the  scientific  world.  It 
was  held  by  the  conservative  majority  that  the  microorganisms  de- 
scribed by  Leeuwenhoek  and  others  after  him  were  produced  by 
spontaneous  generation.  The  doctrine  of  spontaneous  generation,  in 
fact,  was  solidly  established  and  sanctified  by  tradition,  and  had  been 
applied  in  the  past  not  alone  to  microorganisms.2  And  it  must  not  be 
forgotten  that  without  the  aid  of  our  modern  methods  of  study,  satis- 
factory proof  for  or  against  such  a  process  was  not  easily  brought. 

Needham,  who  published  in  1749,  had  spent  much  time  in  fortify- 
ing his  opinions  in  favor  of  spontaneous  generation  by  extensive 
experimentation.  He  had  placed  putrefying  material  and  vegetable 
infusions  in  sealed  flasks,  exposing  them  for  a  short  time  to  heat,  by 
immersing  them  in  a  vessel  of  boiling  water,  and  had  later  shown 
them  to  be  teeming  with  microorganisms.  He  was  supported  in  his 
views  by  no  less  an  authority  than  Buffon.  The  work  of  Needham, 
however,  showed  a  number  of  experimental  inaccuracies  which  were 
thoroughly  sifted  by  the  Abbe  Spallanzani.  This  investigator  re- 
peated the  experiments  of  Needham,  employing,  however,  greater 
care  in  sealing  his  flasks,  and  subjecting  them  'to  a  more  thorough 
exposure  to  heat.  His  results  did  not  support  the  views  of  Needham, 
but  were  answered  by  the  latter  with  the  argument  that  by  excessive 
heating  he  had  produced  chemical  changes  in  his  solutions  which  had 
made  spontaneous  generation  impossible. 

The  experiments  of  Schulze,  in  1836,  who  failed  to  find  living 
organisms  in  infusions  which  had  been  boiled,  and  to  which  air  had 
been  admitted  only  after  passage  through  strongly  acid  solutions, 
and  similar  results  obtained  by  Schwann,  who  had  passed  the  air 
through  highly  heated  tubes,  were  open  to  criticism  by  their  oppo- 
nents, who  claimed  that  chemical  alteration  of  the  air  subjected  to 
such  drastic  influences  had  been  responsible  for  the  absence  of  bac- 
teria in  the  infusion.  Similar  experiments  by  Schroeder  and  Dusch, 
who  had  stoppered  their  flasks  with  cotton  plugs,  were  not  open  to 


2  Valleri-Radot,  in  his  life  of  Pasteur,  states  that  Van  Helmont,  in  the  six- 
teenth century,  had  given  a  celebrated  prescription  for  the  creation  of  mice 
from  dirty  linen  and  a  few  grains  of  wheat  or  pieces  of  cheese.  During  the  centu- 
ries following,  although,  of  course,  such  remarkable  and  amusing  beliefs  no  longer 
held  sway,  nevertheless  the  question  of  spontaneous  generation  of  minute  and 
structureless  bodies,  like  the  bacteria,  still  found  learned  and  thoughtful  partisans. 


DEVELOPMENT  AND  SCOPE  OF  BACTERIOLOGY  5 

this  objection,  but  had  also  failed  to  convince.  The  question  was  not 
definitely  settled  until  the  years  immediately  following  1860,  when 
Pasteur  conducted  a  series  of  experiments  which  were  not  only  im- 
portant in  incontrovertibly  refuting  the  doctrine  of  spontaneous 
generation,  but  in  establishing  the  principles  of  scientific  investiga- 
tion which  have  influenced  bacteriological  research  since  his  time.3 

Pasteur  attacked  the  problem  from  two  points  of  view.  In  the 
first  place  he  demonstrated  that  when  air  was  filtered  through  cotton- 
wool innumerable  microorganisms  were  deposited  upon  the  filter.  A 
single  shred  of  such  a  contaminated  filter  dropped  into  a  flask  of  pre- 
viously sterilized  nutritive  fluid  sufficed  to  bring  about  a  rapid  and 
luxuriant  growth  of  microorganisms.  In  the  second  place,  he  suc- 
ceeded in  showing  that  similar,  sterilized  ' '  putrescible "  liquids,  if 
left  in  contact  with  air,  would  remain  uncontaminated  provided  that 
the  entrance  of  dust  particles  were  prohibited.  This  he  succeeded  in 
doing  by  devising  flasks,  the  necks  of  which  had  been  drawn  out  into 
fine  tubes  bent  in  the  form  of  a  U.  The  ends  of  these  U-tubes,  being 
left  open,  permitted  the  sedimentation  of  dust  from  the  air  as  far  as 
the  lowest  angle  of  the  tube,  but,  in  the  absence  of  an  air  current, 
no  dust  was  carried  up  the  second  arm  into  the  liquid.  In  such  flasks, 
he  showed  that  no  contamination  took  place  but  could  be  immedi- 
ately induced  by  slanting  the  entire  apparatus  until  the  liquid  was 
allowed  to  run  into  the  bent  arm  of  the  U-tube.  Finally,  by  exposing 
a  series  of  flasks  containing  sterile  yeast  infusion,  at  different  atmos- 
pheric levels,  in  places  in  which  the  air  was  subject  to  varying 
degrees  of  dust  contamination,  he  showed  an  inverse  relationship 
between  the  purity  of  the  air  and  the  contamination  of  his  flasks 
with  microorganisms. 

The  doctrine  of  spontaneous  generation  had  thus  received  its 
final  refutation,  except  in  one  particular.  It  was  not  yet  clear  why 
complete  sterility  was  not  always  obtained  by  the  application  of 
definite  degrees  of  heat.  This  final  link  in  the  chain  of  evidence  was 
supplied,  some  ten  years  later,  by  Cohn,  who,  in  1871,  was  the  first  to 


3  In  a  letter  to  his  foremost  opponent,  at  this  period,  Pasteur  writes:  "In 
experimental  science,  it  is  always  a  mistake  not  to  doubt  when  facts  do  not  compel 
affirmation. ' ' 

The  critical  spirit  pervading  the  scientific  thought  of  that  time  in  France  is 
also  well  expressed  by  Oliver  Wendell  Holmes,  who  said  that  he  had  learned  three 
things  in  Paris:  "Not  to  take  authority  when  I  can  have  facts,  not  to  guess  when 
I  can  know,  and  not  to  think  that  a  man  must  take  physic  because  he  is  sick. ' ' 


6  BIOLOGY  AND  TECHNIQUE 

observe  and  correctly  interpret  bacterial  spores  and  to  demonstrate 
their  high  powers  of  resistance  against  heat  and  other  deleterious 
influences. 

Meanwhile,  Pasteur,  parallel  with  his  researches  upon  sponta- 
neous generation,  had  been  carrying  on  experiments  upon  the  subject 
of  fermentation  along  the  lines  suggested  by  Cagniard-Latour.  As  a 
consequence  of  these  experiments,  he  not  only  confirmed  the  opinions 
both  of  this  author  and  of  Schwann  concerning  the  fermentation  of 
beer  and  wine  by  yeasts,  but  was  able  to  show  that  a  number  of 
other  fermentations,  such  as  those  of  lactic  and  butyric  acid,  as  well 
as  the  decomposition  of  organic  matter  by  putrefaction,  were  directly 
due  to  the  action  of  microorganisms.  It  was  the  discovery  of  the 
living  agents  underlying  putrefaction,  especially,  which  exerted  the 
most  active  influence  upon  the  medical  research  of  the  day.  This  is 
illustrated  by  Lister's  work.  The  suppurative  processes  occurring 
in  infected  wounds  had  long  been  regarded  as  a  species  of  putrefac- 
tion, and  Lord  Lister,  working  directly  upon  the  premises  supplied 
by  Pasteur,  introduced  into  both  the  active  and  prophylactic  treat- 
ment of  surgical  wounds  the  antiseptic  principles  which  alone  have 
made  modern  surgery  possible. 

There  now  followed  a  period  in  which  bacteriological  investiga- 
tion was  concentrated  upon  problems  of  etiology.  Stimulated  by 
Pasteur's  successes,  the  long-cherished  hope  of  finding  some  specific 
microorganism  as  the  causal  agent  in  each  infectious  disease  was 
revived. 

Pollender,  in  1855,  had  reported  the  presence  of  rod-shaped  bodies 
in  the  blood  and  spleen  of  animals  dead  of  anthrax.  Brauell,  several 
years  later,  had  made  similar  observations  and  had  expressed  definite 
opinions  as  to  the  causative  relationship  of  these  rods  to  the  disease. 
Convincing  proof,  however,  had  not  been  brought  by  either  of  these 
observers.  Finally,  in  1863,  Davaine,  in  a  series  of  brilliant  investi- 
gations, not  only  confirmed  the  observations  of  the  two  authors 
mentioned  above,  but  succeeded  in  demonstrating  that  the  disease 
could  be  transmitted  by  means  of  blood  containing  these  rods  and 
could  never  be  transmitted  by  blood  from  which  these  rods  were 
absent.  Anthrax,  thus,  is  the  first  disease  in  which  definite  proof  of 
bacterial  causation  was  brought. 

Speaking  before  the  French  Academy  of  Medicine  at  this  time, 
Davaine  suggested  that  the  manifestations  of  the  disease  might  in 
reality  represent  the  results  of  a  specific  fermentation  produced  by 


DEVELOPMENT  AND   SCOPE   OF   BACTERIOLOGY  7 

the  bacilli  he  had  found.  This,  in  a  crude  way,  expresses  the  modern 
conception  of  infectious  disease. 

Within  a  few  years  after  this,  1868,  the  adherents  of  the  parasitic 
theory  of  infectious  diseases  were  further  encouraged  by  the  dis- 
covery, by  Obermeier,  of  a  spirillum  in  the  blood  of  patients  suffering 
from  relapsing  fever.  It  is  not  surprising  that  the  successes  attained 
in  these  diseases,  fostering  hope  of  analogous  results  in  all  other 
similar  conditions,  but  without  the  aid  of  adequate  experimental 
methods,  should  have  led  to  many  unjustified  claims  and  to  much 
fantastic  theorizing.  Thus  Hallier,  at  about  this  time,  advanced  a 
theory  as  to  the  etiology  of  infectious  diseases,  in  which  he  attrib- 
uted all  such  conditions  to  the  molds  or  hyphomycetes,  regarding 
the  smaller  form  or  bacteria  as  developmental  stages  of  .these  more 
complicated  forms.  Extravagant  conjectures  of  this  kind,  however, 
did  not  maintain  themselves  for  any  length  of  time  in  the  light  of  the 
critical  attitude  which  was  already  pervading  bacteriological  re- 
search. 

Progress  was  made  during  the  years  immediately  following, 
chiefly  in  the  elucidation  of  suppurative  processes.  Rindfleisch,  von 
Recklinghausen,  and  Waldeyer,  almost  simultaneously,  described 
bodies  which  they  observed  in  sections  of  tissue  containing  abscesses, 
and  which  they  believed  to  be  microorganisms.  Notable  support  was 
given  to  their  opinion  by  similar  observations  made  upon  pus  by 
Klebs,  in  1870.  In  view,  however,  of  the  purely  morphological  nature 
of  their  studies,  the  opinions  of  these  observers  did  not  entirely  pre- 
vail. Satisfactory  methods  of  cultivation  and  isolation  had  not  yet 
been  developed,  and  Billroth  and  his  followers,  with  a  conservatism 
entirely  justified  under  existing  conditions,  while  admitting  the  con- 
stant presence  of  bacteria  in  purulent  lesions,  denied  their  etiological 
significance.  The  controversy  that  followed  was  rich  in  suggestions 
which  greatly  facilitated  the  work  of  later  investigators,  but  could 
not  be  definitely  settled  until  1880,  when  Koch  introduced  the  tech- 
nical methods  which  have  made  bacteriology  an  exact  science.  By 
the  use  of  solid  nutritive  media,  the  isolation  of  bacteria  and  their 
biological  study  in  pure  culture  were  made  possible.  At  about  the 
same  time  the  use  of  anilin  dyes,  developed  by  Weigert,  Koch,  and 
Ehrlich,  was  introduced  into  morphological  study  and  facilitated  the 
observation  of  the  finer  structural  details  which  had  been  unnoticed 
while  only  the  grosser  methods  employed  for  tissue  staining  had  been 
available. 


8  BIOLOGY  AND  TECHNIQUE 

With  the  publication  of  Koch's  work,  there  began  an  era  un- 
usually rich  in  results  held  in  leash  heretofore  by  inadequate  tech- 
nical methods.  The  discovery  of  the  typhoid  bacillus  in  1880,  of  the 
bacillus  of  fowl  cholera  and  the  pneumococcus  in  the  same  year,  and 
of  the  tubercle  bacillus  in  1882,  initiated  a  series  of  etiological  dis- 
coveries which,  extending  over  not  more  than  fifteen  years,  eluci- 
dated the  causation  of  a  majority  of  the  infectious  diseases. 

Coincident  with  the  elucidation  of  etiological  facts  began  the 
inquiry  into  that  field  which  is  now  spoken  of  as  the  science  of 
immunity.  The  phenomena  which  accompany  the  development  of 
insusceptibility  to  bacterial  infections  in  man  and  in  animals,  first 
studied  by  Pasteur,  have  become  the  subject  of  innumerable  re- 
searches and  have  led  to  results  of  the  utmost  practical  value. 

The  problems  which  were  encountered  were  first  studied  from  a 
purely  bacteriological  point  of  view,  but  their  solution  has  shed  light 
upon  biological  principles  of  the  broadest  application.  Investigations 
into  the  properties  of  immune  sera,  while  making  bacteriology  one  of 
the  most  important  branches  of  diagnostic  and  therapeutic  medicine, 
have,  at  the  same  time,  inseparably  linked  it  with  physiology  and 
experimental  pathology. 

By  the  revelations  of  etiological  research,  and  by  the  study  of  the 
biological  properties  of  pathogenic  bacteria,  contagion,  an  enemy 
hitherto  unseen  and  mysterious,  was  unmasked,  and  rational  cam- 
paigns of  public  sanitation  and  personal  hygiene  were  made  possible. 
Upon  the  same  elucidations  has  depended  the  development  of  modern 
surgery — a  science  which  without  asepsis  and  antisepsis  would  have 
been  doomed  to  remain  in  its  medieval  condition. 

Apart  from  its  importance  in  the  purely  medical  sciences,  the 
study  of  the  bacteria  has  shed  beneficial  light,  moreover,  upon  many 
other  fields  of  human  activity.  In  their  relationship  to  decomposi- 
tion, the  conditions  of  the  soil,  and  to  diseases  of  plants,  the  bacteria 
have  been  found  to  occupy  a  condition  of  great  importance  in  agri- 
culture. Knowledge  of  bacterial  and  yeast  ferments,  furthermore, 
has  become  the  scientific  basis  of  many  industries,  chiefly  those  con- 
cerned in  the  production  of  wine,  beer,  and  dairy  products. 

The  scope  of  bacteriology  is  thus  a  wide  one,  and  none  of  its 
various  fields  has,  as  yet,  been  fully  explored.  The  future  of  the 
science  is  rich  in  allurement  of  interest,  in  promise  of  result,  and  in 
possible  benefit  to  mankind. 


CHAPTER  II 

GENERAL    MORPHOLOGY,    REPRODUCTION,    AND    CHEMICAL 
AND   PHYSICAL  PROPERTIES   OF   THE   BACTERIA 

BACTERIA  are  exceedingly  minute  unicellular  organisms  which  may 
occur  perfectly  free  and  singular,  or  in  larger  or  smaller  aggregations, 
thus  forming  multicellular  groups  or  colonies,  the  individuals  of  which 
are,  however,  physiologically  independent. 

The  cells  themselves  have  a  number  of  basic  or  ground  shapes  which 
may  be  roughly  considered  in  three  main  classes :  The  cocci  or  spheres, 
the  bacilli  or  straight  rods,  and  the  spirilla  or  curved  rod  forms. 

The  cocci  are,  when  fully  developed  and  free,  perfectly  spherical. 
When  two  or  more  are  in  apposition,  they  may  be  slightly  flattened 
along  the  tangential  surface,  giving  an  oval  appearance. 

The  bacilli,  or  rod-shaped  forms,  consist  of  elongated  cells  whose 
long  diameter  may  be  from  two  to  ten  times  as  great  as  their  width, 
with  ends  squarely  cut  off,  as  in  the  case  of  bacillus  anthracis,  or  gently 
rounded  as  in  the  case  of  the  typhoid  bacillus. 

The  spirilla  may  vary  from  small  comma-shaped  microorganisms, 
containing  but  a  single  curve,  to  longer  or  more  sinuous  forms  which 
may  roughly  be  compared  to  a  corkscrew,  being  made  up  of  five,  six 
or  more  curves.  The  turns  in  the  typical  microorganisms  of  this  class 
are  always  in  three  planes  and  are  spiral  rather  than  simply  curved. 

Among  the  known  microorganisms,  the  bacilli  by  far  outnumber 
other  forms,  and  are  probably  the  most  common  variety  of  bacteria  in 
existence.  Many  variations  from  these  fundamental  types  may  occur 
even  under  normal  conditions,  but  contrary  to  earlier  opinions  it  is 
now  positively  known  that  cocci  regularly  reproduce  cocci,  bacilli 
bacilli,  and  spirilla  spirilla,  there  being,  as  far  as  we  know,  no.  mutation 
from  one  form  into  another. 

The  size  of  bacteria  is  subject  to  considerable  variation.  Cocci  may 
vary  from  .15  ;u  to  2.  ju  in  diameter.  The  average  size  of  the  ordinary 
pus  coccus  varies  from  .8  p.  to  1.2  /x  in  diameter.  Fischer  has  given  a 
graphic  illustration  of  the  size  of  a  staphyloccocus  by  calculating  that 
one  billion  micrococci  could  easily  be  contained  in  a  drop  of  water  hav- 

9 


10  BIOLOGY  AND  TECHNIQUE 

ing  a  volume  of  one  cubic  millimeter.  Among  the  bacilli  the  range  of 
size  is  subject  to  even  greater  variations.  Probably  the  smallest  of 
the  common  bacilli  is  the  bacillus  of  influenza,  which  measures  about 
.5  fji  in  length  by  .2 /*  in  thickness.  The  limit  o!'  the  optical  possi- 
bilities of  the  modern  microscope  is  almost  reached  by  some  of  the 
known  microorganisms,  and  there  are  some  diseases  which  are  caused 
by  organisms  so  small  as  to  be  invisible  by  any  of  our  present 
methods.  In  fact,  the  virus  causing  the  peripneumonia  of  cattle  has 
been  shown  to  pass  through  the  pores  of  a  Berkefeld  filter,  which 
are  impenetrable  to  the  smallest  of  the  known  bacteria.1  The  coc- 
coid  or  globoid  bodies  grown  by  Noguchi  from  poliomyelitis  virus 
are  small  enough  to  pass  through  such  filters,  but  are  still  visible  with 
the  highest  lens  magnifications.  Whether  or  not  these  minute  bodies 
should  be  regarded  as  bacteria  is  questionable.  It  seems  likely  that 
they  represent  an  entirely  different  class  of  organisms.  It  is  worth 


FIG.  1. — TYPES  OF  BACTERIAL  MORPHOLOGY. 

mentioning,  also,  that  organisms  like  streptococci  may  show  minute 
forms,  especially  when  grown  under  anaerobic  conditions,  which  are 
almost  as  small  as  the  globoid  bodies.  It  seems  a  general  rule  that 
anaerobically  grown  cocci  may  assume  smaller  forms. 

MORPHOLOGY  OF  THE  BACTERIAL  CELL. — When  unstained,  most 
bacteria  are  transparent,  colorless,  and  apparently  homogeneous 
bodies  with  a  low  refractive  index.  The  cells  themselves  consist  of 
a  mass  of  protoplasm,  surrounded,  in  most  instances,  by  a  delicate 
cell  membrane. 

The  presence  of  a  nucleus2  in  bacterial  cells,  though  denied  by 
the  earlier  writers,  has  been  demonstrated  beyond  question  by  Zett- 
now,  Nakanishi,3  and  others.  The  original  opinion  of  Zettnow  was 
that  the  entire  bacterial  body  consisted  of  nuclear  material  inti- 

1  Nocard  and  Eoux,  Ann.  Past.,  12,  1898. 

2  A.  Fischer,  Jahrbiicher  f .  wissen.  Botanik,  xxvii. 

3  Nakatnishi,  Munch,  med.  Woch.,  vi,  1900. 


MORPHOLOGY,  REPRODUCTION,  ETC.  11 

matcly  intermingled  with  the  cytoplasm.  The  opinion  now  held  by 
most  observers  who  have  studied  this  phase  of  the  subject  favors 
the  existence  of  an  ectoplasmic  zone  which  includes  cell  membrane 
and  flagella,  but  is  definitely  a  part  of  the  cytoplasm,  and  an  ento- 
plasm  in  which  is  concentrated  the  nuclear  material.  Biitschli* 
claims  to  have  demonstrated  within  this  entoplasmic  substance  a 
reticular  meshwork,  between  the  spaces  of  which  lie  granules  of 
chromophilic  or  nuclear  material.  Confirmation  of  this  opinion  has 
been  brought  by  Zettnow5  and  others.  Nakanishi,  working  with  a 
.special  staining  method,  asserts  that  some  microorganisms  show 
within  the  entoplasmic  zone  a  well-defined,  minute,  round  or  oval 
nucleus,  which  possesses  a  definitely  characteristic  staining  reaction.6 
In  the  bodies  of  a  large  number  of  bacteria,  notably  in  those  of 
the  diphtheria  group,  Ernst,7  Babes,8  and  others  have  demonstrated 
granular,  deeply  staining  bodies  now  spoken  of  as  met  achromatic 
granules,  or  Babes-Ernst  granules,  or,  because  of  their  frequent  posi- 
tion at  the  ends  of  bacilli,  as  polar  bodies.  These  structures  are 
irregular  in  size  and  number,  and  have  a  strong  affinity  for  dyes. 
They  are  stained  distinctly  dark  in  contrast  to  the  rest  of  the  bac- 
terial cell  with  methylene  blue,  and  may  be  demonstrated  by  the 
special  methods  of  Neisser  and  of  Roux.9  Their  interpretation  has 
been  a  matter  of  much  difficulty  and  of  varied  opinion.  Those  who 
first  observed  them  held  that  they  were  a  part  of  the  nuclear  material 
of  the  cell.  Others  have  regarded  them  as  an  early  stage  in  spore 
formation,  or  as  arthrospores.10  Again,  they  have  been  interpreted 
as  structures  comparable  to  the  centrosomes  of  other  unicellular 


*Biitschli,  «Bau  der  Bakterien,"  Leipzig,  1890. 

5  Zettnow,  Zeit.  f.  Hyg.,  xxiv,  1897. 

'The  method  of  Nakanishi  is  carried  out  as  follows:  Thoroughly  cleansed 
slides  are  covered  with  a  saturated  aqueous  solution  of  methylene  blue.  This  is 
spread  over  the  slide  in  an  even  film  and  allowed  to  dry.  After  drying,  the  slide 
should  be  of  a  transparent,  sky-blue  color.  The  microorganisms  to  be  examined  are 
then  emulsified  in  warm  water,  or  are  taken  from  the  fluid  media,  and  dropped  upon 
a  cover  slip.  This  is  placed,  face  downward,  upon  the  blue  ground  of  the  slide.  In 
this  way,  bacteria  are  stained  without  fixation.  Nakanishi  claims  that  by  this 
method  the  entoplasm  is  stained  blue,  while  the  nuclear  material  appears  of  a  red- 
dish or  purplish  hue. 

1  Ernxt,  Zeit.  f.  Hyg.,  iv,  1888. 

8  See  section  on  stains,  p.  — . 

9  Babes,  Zeit.  f .  Hyg.,  v,  1889. 

10  See  section  on  sporulation,  p.  16. 


12  BIOLOGY  AND  TECHNIQUE 

forms.  As  a  matter  of  fact,  the  true  nature  of  these  bodies  is  by  no 
means  certain.  They  are  present  most  regularly  in  microorganisms 
taken  from  young  and  vigorous  cultures  or  in  those  taken  directly 
from  the  lesions  of  disease.  It  is  unlikely  that  they  represent  struc- 
tures in  any  way  comparable  to  spores,  since  cultures  containing 
individuals  showing  metachromatic  granules  are  not  more  resistant 
to  deleterious  influences  than  are  others.  Their  abundant  presence 
in  young  vigorous  cultures  may  indicate  a  relationship  between 
them  and  the  growth  energy  of  the  microorganisms.  There  is  no 
proof,  however,  that  these  bodies  affect  the  virulence  of  the  bacteria. 
Cell  Membrane  and  Capsule. — Actual  proof  of  the  existence  of  a 
cell  membrane  has  been  brought  in  the  cases  of  some  of  the  larger 


y^_.  .,  ;  <fc        M 

FIG.  2. — BACTERIAL  CAPSULES. 

forms  only,11  but  the  presence  of  such  envelopes  may  be  inferred  for 
most  bacteria  by  their  behavior  during  plasmolysis,  where  definite 
retraction  of  the  protoplasm  from  a  well-defined  cell  outline  has  been 
repeatedly  observed.  The  occurrence,  furthermore,  of  so-called 
" shadow  forms"  which  appear  as  empty  capsules,  and  of,  occasion- 
ally, a  well-outlined  cell  body,  after  the  vegetative  form  has  entirely 
degenerated  in  the  course  of  sporulation,  make  the  assumption  of 
the  presence  of  a  cell  membrane  appear  extremely  well  founded. 
Differing  from  the  cell  membranes  of  plant  cells,  cellulose  has  not, 
except  in  isolated  instances,  been  demonstrable  for  bacteria,  and 
the  membrane  is  possibly  to  be  regarded  rather  as  a  peripheral  pro- 
toplasmic zone,  which  remains  unstained  by  the  usual  manipulations. 

llButschli,  loc.  cit. 


MORPHOLOGY,   REPRODUCTION,  ETC  13 

Zettnow,12  who  has  carefully  studied  the  structure  of  some  of  the 
larger  forms,  takes  the  latter  view,  and  regards  the  ' '  ectoplasmic " 
zone  as  a  part  of  the  cell  protoplasm  devoid  of  nuclear  material. 
Zettnow's  opinion  is  borne  out  by  the  greatly  increased  size  of  the 
bacterial  cells  as  seen  by  means  of  special  stains. 

Many  bacteria  have  been  shown  to  possess  a  mucoid  or  gelatinous 
envelope  or  capsule.  According  to  Migula,13  such  an  envelope  is 
present  on  all  bacteria,  though  it  is  in  only  a  few  species  that  it  is 
sufficiently  well  developed  and  stable  to  be  easily  demonstrable  and 
of  differential  value.  When  stained,  the  capsule  takes  the  ordinary 
anilin  dyes  less  deeply  than  does  the  bacterial  cell  body,  and  varies 
greatly  in  thickness,  ranging  from  a  thin,  just  visible  margin  to 
dimensions  four  or  five  times  exceeding  the  actual  size  of  the  bac- 
terial body  itself.  This  structure  is  perfectly  developed  in  a  limited 
number  of  bacteria  only  in  which  it  then  becomes  an  important  aid 
to  identification.  Most  prominent  among  such  bacteria  are  Diplo- 
coccus  pneumonias,  Micrococcus  tetragenus,  the  bacilli  of  the  Fried- 
lander  group,  and  B.  aerogenes  capsulatus.  The  development  of  the 
capsule  seems  to  depend  intimately  upon  the  environment  from 
which  the  bacteria  are  taken.  It  is  most  easily  demonstrable  in 
preparations  of  bacteria  taken  directly  from  animal  tissues  and 
fluids,  or  from  media  containing  animal  serum  or  milk.  If  cultivated 
for  a  prolonged  period  upon  artificial  media,  many  otherwise  capsu- 
lated  microorganisms  no  longer  show  this  characteristic  structure. 

Capsules  may  be  demonstrated  on  bacteria  taken  from  artificial 
media  most  successfully  when  albuminous  substances,  such  as  ascitic 
fluid  or  blood  serum,  are  present  in  the  culture  media,  or  when  the 
bacteria  are  smeared  upon  cover  slip  or  slide  in  a  drop  of  beef  or 
other  serum.14  Most  observers  believe  that  the  capsule  represents  a 
swelling  of  the  ectoplasmic  zone  of  bacteria.  By  others  it  is  regarded 
as  an  evidence  of  the  formation  of  a  mucoid  intercellular  substance, 
some  of  which  remains  adherent  to  the  individual  bacteria  when 
removed  from  cultures.  It  is  noticeable,  indeed,  that  some  of  the 
capsulated  bacteria,  especially  Streptococcus  mucosus  and  B.  muco- 
sus  capsulatus,  develop  such  slimy  and  gelatinous  colonies  that,  when 
these  are  touched  with  a  platinum  wire,  mucoid  threads  and  strings 


12  Zettnow,  loc.  cit. 

13 Migula,  "Systeme  d.  Bakterien,"  1,  p.  56. 

14  Hiss,  Jour.  Exp.  Med.,  vi,  1905. 


14  BIOLOGY  AND  TECHNIQUE 

adhere  to  the  loop.     Exactly  what  the  significance  of  the  capsules 
is  cannot  yet  be  decided. 

There  is,  however,  definite  reason  to  believe  that  there  is  a  direct 
relation  between  virulence  and  capsulation ;  capsulated  bacteria  are 
less  easily  taken  up  by  phagocytes  than  are  the  iion-capsulated  mem- 
bers of  the  same  species.  Also,  as  Porges  and  others  have  shown, 
capsulated  organisms  are  not  easily  amenable  to  the  agglutinating 
action  of  immune  sera.  Many  bacteria  (plague,  anthrax)  which  are 
habitually  uncapsulated  on  artificial  media  acquire  capsules  within 
the  infected  animal  body.  Also  in  some  species  (pneumococci),  the 
loss  of  capsule  formation  as  cultivated  on  the  simpler  media  is  accom- 
panied by  a  diminution  of  virulence. 

Organs  of  Locomotion. — When  suspended  in  a  drop  of  fluid  many 
bacteria  are  seen  to  be  actively  motile.  It  is  important,  however,  in 
all  cases  to  distinguish  between  actual  motility  and  the  so-called 
Brownian  or  molecular  movement  which  takes  place  whenever  small 
particles  are  held  in  suspension  in  a  fluid. 

Brownian  or  molecular  movement  is  a  phenomenon  entirely  ex- 
plained by  the  physical  principles  of  surface  tension,  and  has  abso- 
lutely no  relation  to  independent  motility.  It  may  be  seen  when 
particles  of  carmine  or  any  other  insoluble  substance  are  suspended 
in  water,  and  consists  in  a  rapid  to  and  fro  vacillation  during  which 
there  is  actually  no  permanent  change  in  position  of  the  moving  par- 
ticle except  inasmuch  as  this  is  influenced  by  currents  in  the  drop. 

The  true  motility  of  bacteria,  on  the  other  hand,  is  active  motion 
due  to  impulses  originating  in  the  bacteria  themselves,  where  the 
actual  position  of  the  bacterium  in  the  field  is  permanently  changed. 

The  ability  to  move  in  this  way  is,  so  far  as  we  know,  limited 
almost  entirely  to  the  bacilli  and  spirilla,  there  being  but  few  in- 
stances where  members  of  the  coccus  group  show  active  motility.  In 
all  cases,  with  the  exception  of  some  of  the  spirochetes,  where  mo- 
tility may  occasionally  be  due  to  an  undulating  membrane  margin- 
ally placed  along  the  body,  bacterial  motility  is  due  to  hair-like 
organs  known  as  flagella.  These  flagella  have  rarely  been  seen  during 
life,  and  their  recognition  and  study  has  been  made  possible  only  by 
special  staining  methods,  such  as  those  devised  by  Loeffler,  van 
Ermengem,  Pitt,  and  others. 

In  such  stained  preparations,  the  bacterial  cell  bodies  often 
appear  thicker  than  when  ordinary  dyes  are  used,  and  the  flagella 
apparently  are  seen  to  arise  from  the  thickened  ectoplasmic  zone. 


MORPHOLOGY,  REPRODUCTION,  ETC.  15 

The  flagella  are  long  filaments,  averaging  in  thickness  from  one- 
tenth  to  one-thirtieth  that  of  the  bacterial  body,  which  often  are 
delicately  waved  and  undulating,  and,  judging  from  the  positions  in 
which  they  become  fixed  in  preparations,  move  by  a  wavy  or  screw- 
like  motion.  In  length  they  are  subject  to  much  variation,  but  are 
supposed  to  be  generally  longer  in  old  than  in  young  cultures.  Very 
short  flagella  have  been  described  only  on  nitrosomonas,  one  of  the 
nitrifying  bacteria  discovered  by  Winogradsky.15 

As  to  the  finer  structures  of  flagella,  little  can  be  made  out  except 
that  they  possess  a  higher  refractive  index  than  the  cell  body  itself, 
and  that  they  can  be  stained  only  with  those  dyes  which  bring  clearly 
into  view  the  supposedly  true  cytoplasm  of  the  cell.  Whether  they 
penetrate  this  cytoplasmic  membrane  or  whether  they  are  a  direct 
continuation  of  this  peripheral  zone  of  the  bacterial  body,  can  not  be 
decided. 

The  manner  in  which  bacteria  move  is  naturally  subject  to  some 


FIG.  3. — ARRANGEMENT  OF  BACTERIAL  FLAGELLA. 

variation  depending  upon  the  number  and  position  of  the  flagella 
possessed  by  them.  Whether  bacteria  exercise  or  not  the  power  of 
motility  depends  to  a  large  extent  upon  their  present  or  previous 
environment.  They  are  usually  most  motile  in  vigorous  young  cul- 
tures of  from  twenty-four  to  forty-eight  hours'  growth  in  favorable 
media.  In  old  cultures  motility  may  be  diminished  or  even  inhibited 
by  acid  formation  or  by  other  deleterious  products  of  the  bacterial 
metabolism. 

At  the  optimum  growth-temperature  motility  is  most  active,  and 
a  diminution  or  increase  of  the  temperature  to  any  considerable  de- 
gree diminishes  or  inhibits  it.  Thus  actively  motile  organisms,  in  the 
fluid  drop,  may  be  seen  to  diminish  distinctly  in  activity  when  left 
for  any  prolonged  time  in  a  cold  room,  or  when  the  preparation  is 
chilled.  Any  influence,  in  other  words,  chemical  or  physical,  which 

15  Winogradsky,  Arch.  <lcs  sci.  biologiques,  St.  Petersburg,  1892,  I,  1  and  2. 


16  BIOLOGY  AND  TECHNIQUE 

tends  to  injure  or  depress  physiologically  the  bacteria  in  any  way,  at 
the  same  time  tends  to  inhibit  their  motility. 

Messea 18  has  proposed  a  classification  of  bacteria  which  is  based 
upon  the  arrangement  of  their  organs  of  motility,  as  follows : 

I.  Gymnobacteria,  possessing  no  flagella. 
II.  Trichobacteria,  with  flagella. 

1.  Monotricha,  having  a  single  flagellum  at  one  pole. 

2.  Lophotricha,  having  a  tuft  of  flagella  at  one  pole. 

3.  Amphitricha,  with  flagella  at  both  poles. 

4.  Peritricha,  with  flagella  completely  surrounding  the  bac- 

terial body. 

Bacterial  Spores. — A  large  number  of  bacteria  possesses  the  power 
of  developing  into  a  sort  of  encysted  or  resting  stage  by  a  process 
commonly  spoken  of  as  sporulation  or  spore  formation.  The  forma- 
tion of  spores  by  bacteria  depends  largely  upon  environmental  con- 
ditions, and  the  optimum  environment  for  spore  formation  differs 
greatly  for  various  species.  It  is  usually  necessary  that  a  tempera- 
ture of  over  20°  C.  exist  in  order  that  spores  may  be  formed.  Un- 
favorable factors,  like  acid  formation,  accumulation  of  bacterial 
products  in  old  cultures,  or  lack  of  nutrition,  frequently  seem  to  con- 
stitute the  stimuli  which  lead  to  sporulation.  In  the  case  of  some 
species,  notably  the  anthrax  bacillus,  spores  are  formed  only  in  the 
presence  of  free  oxygen  and  are  therefore  never  formed  within  the 
tissues  of  infected  animals.  It  is  claimed  that  some  of  the  pathogenic 
anaerobes,  like  B.  tetani  and  the  bacillus  of  malignant  edema,  may 
form  spores  anaerobically.  Nevertheless  it  has  been  observed  that 
when  an  absolute  exclusion  of  oxygen  is  practiced  in  the  cultivation 
of  these  bacteria,  vegetative  forms  only  are  seen  in  the  cultures.17 

The  process  of  sporulation  is  by  no  means  to  be  regarded  as  a 
method  of  multiplication,  since  it  rarely  occurs  that  a  single  bacillus 
produces  more  than  one  spore.  In  some  species  of  bacteria  the  for- 
mation of  several  spores  in  one  individual  has  occasionally  been  ob- 
served, but  there  can  be  no  question  about  the  fact  that  such  a 
condition  is  exceptional. 

Varieties  of  spores  are  often  recognized,  the  so-called  arthro- 
spores  and  the  true  spores  or  endospores.  It  is  seriously  in  doubt 
whether  the  structures  once  spoken  of  as  arthrospores  should  be 


16  Messea,  Cent.  f.  Bakt,,  I,  Eef.  ix,  1891. 

17  Zinsser,  Jour.  Exp.  Med.,  viii,  1906,  p.  542 


MORPHOLOGY,  REPRODUCTION,  ETC.  17 

considered  as  in  any  way  comparable  to  true  spores.  They  are  rep- 
resented by  the  granular  and  globular  appearances  occasionally  ob- 
served in  old  cultures  of  some  bacteria,  notably  streptococcus,  cholera 
spirillum,  diphtheria  bacillus,  and  others.  It  was  believed  that  they 
were  due  to  a  transformation  of  certain  individuals  of  the  cultures 
into  more  resistant  forms.  It  is  probable,  however,  that  such  struc- 
tures are  merely  to  be  regarded  as  evidences  of  involution  or  degen- 
eration, since  it  has  never  been  demonstrated  that  cultures  contain- 
ing them  are  more  resistant  either  to  disinfectants  or  to  heat,  than 
cultures  showing  no  evidences  of  such  forms.  The  true  spores  or 
endospores  are  most  common  among  bacilli,  and  are  rarely  observed 
among  the  spherical  bacteria.  They  arise  within  the  body  of  the 
individual  bacterium  as  a  small  granule  which  probably  represents  a 
concentration  of  the  protoplasmic  substance.  Nakanishi78  claims 
that  there  is  a  definite  relation  between  these  sporogenic  globules 
and  the  nuclear  material  of  the  bacterial  cell.  At  the  time  at  which 

o  o  m 

FIG.  4. — VARIOUS  POSITIONS  OP  SPORES  IN  BACTERIAL  CELLS. 

sporulation  occurs  there  is  usually  a  slight  and  gradual  thickening 
of  the  bacillary  body.  After  the  formation  of  this  thickening,  a 
spore  membrane  appears  about  the  same  thickened  area.  The  com- 
pleted spore  is  usually  round  or  oval,  has  an  extremely  high  refract- 
ive index,  and  a  membrane  which  is  very  resistant.  Muhlschlegel19 
believes  that  the  spore  membrane  is  a  double  structure,  and,  as  stated 
before,  Nakanishi  believes  that  the  spore  contains  nuclear  material. 

The  position  of  the  spore  in  the  mother  cell  is  of  some  differential 
importance  in  that  it  is  usually  constant  for  one  and  the  same  species. 
Thus,  the  spores  of  the  tetanus  bacillus  are  regularly  situated  at  the 
extreme  ends  of  the  bacillary  bodies,  while  those  of  anthrax  are 
situated  at  or  near  the  middle. 

Physiologically,  sporulation  is  probably  to  be  regarded  as  a 
method  of  encystment  for  the  purpose  of  resisting  unfavorable  envi- 
ronment, and  it  is  indeed  true  that  species  of  bacteria  the  vegetative 

18  Nakanislii,  Munch,  med.  Woch.,  1900,  p.  680. 

19  Muhlschlegel,  Cent,  f .  Bakt.,  II  Abt.,  vi,  1900,  p.  65. 


18  BIOLOGY  AND  TECHNIQUE 

forms  of  which  are  rather  easily  injured  by  heat,  light,  drying,  and 
chemicals  have  a  comparatively  enormous  resistance  1o  these  agents 
after  the  formation  of  spores.  Thus,  while  a  10-per-cent  solution  of 
carbolic'  acid  will  kill  the  vegetative  forms  of  anthrax  bacilli  within 
twenty  minutes,  anthrax  spores  are  able  to  resist  the  same  disinfect- 
ant for  a  long  period  in  a  concentration  of  over  50  per  cent;  and 
while  the  vegetative  forms  of  the  same  bacillus  show  little  more 
resistance  against  moist  heat  than  other  vegetative  forms,  the  spores 
will  withstand  the  action  of  live  steam  for  as  long  as  ten  to  twelve 
minutes  and  more. 

Whenever  the  spores  of  any  microorganism  are  brought  into  an 


a,  O    &O    *W      C^*  C?* 

FIG.  5. — GERMINATION  OF  SPORES. 

environment  suitable  for  bacterial  growth  as  to  temperature,  mois- 
ture, and  nutrition,  the  spores  develop  into  vegetative  forms.  This 
process  differs  according  to  species.  In  general  it  consists  of  an 
elongation  of  the  spore  body  with  a  loss  of  its  highly  refractile  char- 
acter and  resistance  to  staining  fluids.  The  developing  vegetative 
cell  may  now  rupture  and  slip  out  of  the  spore  membrane  at  one  oi 
its  poles,  leaving  the  empty  spore  capsule  still  visible  and  attached 
to  the  bacillary  body.  Again,  a  similar  process  may  take  place  equa- 
torially  instead  of  at  the  pole.  In  other  species  again,  there  may 
be  no  rupture  of  the  spore  membrane  at  all,  the  vegetative  form  aris- 
ing by  gradual  elongation  of  the  spore  and  an  absorption  or  solution 
of  the  membrane  which  is  indicated  by  change  in  staining  reaction, 
Division  by  fission  in  the  ordinary  way  then  ensues. 


MORPHOLOGY,   REPRODUCTION,   ETC.  19 

REPRODUCTION  OF  BACTERIA 

Bacteria  multiply  by  cell  division  or  fission.  A  young  individual 
increases  in  size  up  to  the  limits*  of  the  adult  form,  when,  by  simple 
cleavage,  at  right  angles  to  the  long  axis,  without  any  discoverable 
process  of  mitosis  or  nuclear  changes,  it  divides  into  two  individuals. 
In  spite  of  the  claims  of  various  bacteriologists,  notably  Nakanishi,20 
traceable  analogy  to  the  karyokinesis  of  other  cells  has  not  been 
definitely  established.  In  the  case  of  the  spherical  bacteria  a  slight 
change  to  the  elliptical  form  takes  place  just  before  cleavage  and 
this  cleavage  may  occur  in  one  plane  only,  in  two  planes,  or  in  three 
planes.  According  to  the  limitations  of  cleavage  direction,  the  cocci 
assume  a  chained  appearance  (streptococci),  a  grape-like  appearance 
(staphylococci),  or  an  arrangement  in  packets  or  cubes  having  three 
dimensions  (sarcinae).  In  the  cases  of  bacilli  and  spirilla,  cleavage 
takes  place  in  the  direction  of  the  short  axis.  The  individuals,  after 
cleavage,  may  separate  from  each  other,  or  may  remain  mutually 
coherent.  The  cohesion  after  cleavage  is  pronounced  in  some  species 
of  bacteria  and  slight  in  others,  and,  together  with  the  plane  of 
cleavage,  determines  the  morphology  of  the  cell-groups.  Thus  among 
the  cocci  diplo-  or  double  forms,  long  chains  and  short  chains  may 
arise  and  furnish  a  characteristic  of  great  aid  in  differentiation. 
Similarly  among  the  bacilli  there  are  forms  which  appear  character- 
istically as  single  individuals  and  others  which  form  chains  of  vary- 
ing length. 

The  rate  of  growth  varies  to  a  certain  extent  with  the  species, 
and  also  with  the  favorable  or  unfavorable  character  of  the  environ- 
ment. A  generation,  that  is,  the  time  elapsing  in  the  interval  between 
one  cleavage  and  the  next,  has  been  estimated  by  A.  Fischer21  as 
being  about  twenty  minutes  for  the  cholera  spirillum  and  16-20  min- 
utes for  bacillus  coli  communis,  under  the  most  favorable  conditions. 
The  same  author  has  calculated  that  under  these  conditions  a  single 
cholera  spirillum  would  yield  1600  trillions  in  a  single  day.  Such  a 
multiplication  rate,  however,  is  probably  not  usual  under  natural  or 
even  artificial  conditions,  both  on  account  of  lack  of  nutritive  mate- 
rial and  because  of  inhibition  of  the  growth  of  the  bacteria  by  their 
own  products. 

20  Ndkanishi,  Cent.  f.  Bakt.,  I,  xxx,  1901. 

21  A.  Fischer,  ' '  Vorlesungen  iiber  Bakt.,"  Jena,  1903. 


20  BIOLOGY  AND  TECHNIQUE 

VARIATIONS  OF  BACTERIAL  FORMS 

Variations  from  the  basic  forms  considered  in  the  preceding  sec- 
tion may  occur,  but  are  not  common  among  bacteria  under  normal 
conditions.  Thus  the  formation  of  club  shapes  by  a  thickening  of  the 
bacillary  body  at  one  or  both  ends  has  been  frequently  observed 
among  bacteria  of  the  diphtheria  group,  and  in  the  glanders  bacillus, 
and  an"  irregular  beading  is  not  infrequently  observed  in  tubercle 
bacilli  under  normal  conditions.  Such  pictures  can  not,  in  these 
cases,  be  regarded  as  degeneration  or  involution  forms,  since  they  are 


FIG.  6. — DEGENERATION  FORMS  OF  BACILLUS  DIPHTHERIA.     (After  Zettnow.) 

visible  in  young,  actively  growing  cultures  under  ordinary  condi- 
tions. It  is  a  well-known  fact,  furthermore,  that  the  sizes  and  con- 
tours of  bacteria  may  vary  to  some  extent  according  to  the  medium 
on  which  they  are  grown.  This  may,  to  a  degree,  be  due  to  osmotic 
relations.  On  fluid  media,  for  instance,  many  bacteria  may  appear 
larger  and  of  a  less  dense  consistency  than  do  members  of  the  same 
species  cultivated  upon  solid  media. 

Degeneration  Forms. — When  bacteria  are  grown  under  conditions 
which  are  not  entirely  favorable  for  their  development,  or  when  they 
are  grown  for  a  prolonged  period  upon  artificial  culture  media  with- 
out transplantation,  there  may  occur  variations  which  often  depart 
considerably  from  the  ground  type,  known  as  degeneration  or  invo- 
lution forms.  Thus,  in  the  case  of  the  diphtheria  bacillus,  old  cul- 


MORPHOLOGY,  REPRODUCTION,  ETC.  21 

tures  may  contain  long,  irregularly  beaded  forms  with  broad  expan- 
sions at  the  ends.  In  the  case  of  B.  pestis  the  fact  that  large  numbers 
of  oval,  vacuolated  bodies  in  old  cultures  are  formed  regularly  has 
become  of  differential  value.22  These  degeneration  forms  are  shown 
most  characteristically  when  the  bacteria  are  cultivated  on  agar 
containing  3  to  5  per  cent  NaCl. 

Among  the  cocci,  marked  evidences  of  involution  are  often  seen 
in  cultures  of  the  meningococcus  in  the  form  of  large,  swollen  poorly- 
staining  spheres,  and  in  the  case  of  the  pneumococcus  in  the  so-called 
shadow  forms  which  have  the  appearance  of  empty  capsules.  There 


FIG.  7. — DEGENERATION  FORMS  OF  BACILLUS  PESTIS.     (After  Zettnow.) 

are  few  microorganisms  indeed,  in  which  prolonged  cultivation  on 
artificial  media  or  other  unfavorable  influences  do  not  produce  varia- 
tions from  the  ground  type  which  may  often  make  the  cultures  mor- 
phologically unrecognizable.  In  the  case  of  many  of  the  spirilla 
(spirillum  Milleri,  spirillum  Metchnikovi,  etc.)  the  degeneration 
forms  may  appear  within  so  short  a  time  as  two  or  three  days  after 
transplantation. 

CHEMICAL  CONSTITUENTS. — The  bacterial  cell  has  been  found  to 
contain  proteins,  nucleic  acid,  carbohydrates,  fatty  substances  and 
ash.  The  quantitative  chemical  composition  of  dried  mass  cultures 
of  bacteria,  even  of  the  same  species,  shows  rather  extreme  varia- 


22  Hanlcin  and  Leumann,  Cent,  f .  Bakt.,  I,  xxii,  1897. 


22  BIOLOGY  AND  TECHNIQUE 

tions  in  the  percentage  of  these  substances,  depending  upon  the 
nature  of  the  culture  media.  Thus,  80  to  90  per  cent  is  water,  and 
in  the  dry  residue,  the  protein  content,  estimated  from  the  total 
nitrogen,  varies  roughly  from  50  to  80  per  cent.  Cramer23  has 
shown  that  the  cholera  vibrio,  grown  on  bouillon,  contains  69  per 
cent  of  protein  and  26  per  cent  of  ash,  while  on  Uschinsky's  medium 
the  same  organism  contained  only  36  per  cent  protein,  and  14  per 
cent  of  ash.  It  seems  probable  that  such  variations  may  depend  to 
some  degree  on  the  semi-permeable  nature  of  the  bacterial  cell ;  and 
perhaps  to  an  equal  degree  on  the  fact  that  analyses  of  the  mass 
cultures  necessarily  include  any  insoluble  metabolic  products,  such 
as  pigments,  the  production  of  which  is  known  to  vary  with  the 
nature  of  the  media. 

Analyses  made  by  Kappes24  of  B.  prodigiosus,  and  by  Nencki 
and  Scheffer25  of  some  of  the  putrefactive  bacteria,  may  serve  to 
illustrate  the  approximate  proportions  of  the  substances  making  up 
the  bacterial  body.  The  protein  is  calculated  from  the  total  nitro- 
gen, and  since  the  nucleic  acid  of  the  cell  is  not  taken  into  account 
the  results  are  not  accurate. 

B.  Putrefactive 

prodigiosus  Bacteria 

Water    85.45  per  cent.  83.42  per  cent. 

Proteins 10.33     "       "  13.96     "       " 

Fats    0.7       "       "  1.         "       " 

Ash 1.75     "       "  0.78     "       " 

Residue    1.77     "       "  0.84     "       " 

The  bacterial  protein  is  probably  of  several  types.  A  true  glob- 
ulin has  been  described,  and  coagulable  proteins  have  been  demon- 
strated by  Buchner26  in  the  "Pressaft"  or  juice  obtained  by  sub- 
jecting bacteria  to  mechanical  pressure.  The  presence  of  nuclear 
material,  chromatin,  in  bacteria,  as  shown  by  staining  reactions, 
together  with  general  biological  considerations,  indicate  that  some, 
at  least,  of  the  protein  is  probably  combined  with  nucleic  acid  in 
the  form  of  "nucleins"  or  "nucleoproteins. "  The  presence  of  these 


23  Cramer,  Arch.  f.  Hyg.,  xxii,  1895,  1(57. 

24  Kappes,  Analyse  der  Mossonkulturon,  etc.,  Diss.,  Leipzig,  1889. 

*  Nencld  and  Scheffer,  Jour.  f.  prakt.  Chemie,  new  series,  xix,  1880. 
26  Buchner,  Munch,  med.  Woch.,  xliv,  1897,  299. 


MORPHOLOGY,   REPRODUCTION,   ETC.  23 

substances  was  established  by  Ruppel27  who,  in  an  analysis  of  the 
tubercle  bacillus,  obtained  the  following  values  for  100  grams  of 
the  dried  bacilli: 

Nucleic  acid   8.5  grams. 

(Tubcrculinic  acid) 

Nucleoprotamm    25.5  * ' 

Nucleoprotein    23.  ' ' 

Albuminoids    8.3  ' ' 

(Keratin,    etc.) 

Fat  and  wax 26.5  ' ' 

Ash    9.2  " 

More  recently,  nucleic  acid  and  the  products  of  its  hydrolysis 
have  been  shown  to  occur  in  all  species  of  bacteria  investigated. 
Both  purine  and  pyrimidine  bases  have  been  isolated,  and  it  is 
particularly  interesting  that  both  uracil  and  thymin  have  been  found 
by  Levene 28  in  nucleic  acid  from  the  tubercle  bacillus.  The  former 
is  found  only  in  nucleic  acid  from  plant  cells,  while  the  latter  is 
obtained  only  from  animal  sources.  Jones29  states,  however,  that 
uracil  often  results  from  the  decomposition  of  cytosine  of  plant 
nucleic  acids.  On  the  other  hand,  pentose  has  been  found  as  a  de- 
composition product  of  bacterial  nucleic  acids,  and  since  this  sub- 
stance occurs  only  among  the  cleavage  products  of  yeast  and  other 
plant  nucleic  acids,  it  would  tend  to  establish  the  relationship  of 
bacteria  to  the  vegetable  kingdom.  The  evidence  is,  therefore,  con- 
tradictory, and  it  may  ultimately  be  shown  that  nucleic  acid  of 
bacteria  is  not  identical  with  either  the  animal  or  plant  type. 

The  basic  chemical  nature  of  the  protein  of  bacteria  does  not  seem 
to  differ  from  protein  of  other  kinds.  Complete  analysis  of  the 
amino  acid  components  of  purified  preparations  are  wanting.  How- 
ever, the  presence  of  most  of  the  monoamino  acids,  as  well  as  the 
hexone  bases  and  glutamic  acid  have  been  established  by  the  work  of 
Tamura,30  Leach,31  Wheeler,32  and  others.  Tamura  showed  that  the 
hexone  base  content  of  bacterial  protein  was  the  same,  whether 


27  Buppel,  Zeit.  f.  physiol.  Chemie,  xxvi,  1898,  218. 

28  Levene,  J.  of  Med.  Ees.,  vii,  1904,  251. 

29  Jones,  "Nucleic  Acids,'7  New  York,  1914. 

30  Tamura,  Zeit.  physiol.  Chemie,  Ixxxviii,  1913,  190,  and  Ixxxix,  1914,  289. 
81  Leach,  J.  of  Biol.  Chem.,  i,  1906,  463. 

12  Wheeler,  J.  of  Biol.  Chem.,  vi,  1909,  509. 


24  BIOLOGY  AND  TECHNIQUE 

grown  on  broth  or  on  a  protein-free  synthetic  medium,  and  concludes 
that  bacteria  are  capable  of  building  up  their  own  protein,  independ- 
ent of  the  nature  of  their  food  supply. 

Although  there  is  much  uncertainty  about  the  nature  of  the 
relatively  thermostable  toxic  substances  contained  in  bacteria,  the 
endotoxins,  it  is  probable  that  they  are  also  of  protein  nature. 

The  fats,  which  are  demonstrable  both  by  michrochemical 
methods,  staining  with  Sudan  III,  Scharlach  R.,  and  Osmic  acid, 
and  by  alcohol-ether  extraction,  consist  of  fatty  acids,  true  fats,  and 
in  the  case  of  the  tubercle  bacillus,  at  least,  of  waxy  substances.33 
The  acid-fastness  of  these  organisms  is  probably  due  in  part  to  the 
presence  of  these  lipoidal  constituents,  which  are  present  to  the 
extent  of  20  to  40  per  cent  of  the  dry  weight  of  the  bacteria. 

Cellulose  has  been  stated  to  occur  in  certain  species  of  bacteria, 
but  the  evidence  is  unsatisfactory  and  many  investigators  have  failed 
to  recognize  it.  A  hemicellulose  has  also  been  described  by  Nishi- 
mura34  and  by  Tamura.35  Chitin,  the  shell-like  material  making 
up  the  protective  covering  of  lobsters,  crabs,  insects,  etc.,  seems  to 
have  been  definitely  recognized  in  bacteria,  by  Iwanoff,36  who  ob- 
tained considerable  quantities  of  glucosamine  hydrochloride  from  the 
hydrolysis  of  his  preparation.  The  occurrence  of  chitin  in  bacteria 
would  seem,  again,  to  connect  these  organisms  more  closely  to  the 
animal  than  to  the  vegetable  kingdom,  thus  contradicting  the  rela- 
tion suggested  by  the  split  products  of  the  nucleic  acid. 

Glycogen-like  substances  have  been  demonstrated,  according  to 
A.  Fischer 37  in  B.  subtilis  and  B.  coli.  These  bacteria  stained  a  red- 
dish brown  with  iodin,  and  after  treatment  with  weak  acids  were 
shown  to  contain  dextrose.  Hydrolysis  of  other  bacteria  has  given 
reducing  sugars,  resulting  presumably  from  the  decomposition  of 
mucins  and  perhaps  of  gums. 

The  bacterial  ash  consists  largely  of  phosphates  and  chlorides  of 
potassium,  sodium,  calcium  and  magnesium.  The  phosphate  prob- 
ably occurs  for  the  most  part  as  a  constituent  of  the  nucleic  acid. 
Certain  of  the  higher  bacteria  also  contain  iron  oxide  in  granular 


33  De  Schweints  and  Dorset,  Cent.  f.  Bakt.,  Erste  Abt.,  xxii,  1897,  209. 

34  NisJiimura,  Arch,  f .  Hyg.,  xviii,  1893,  318  and  xxi,  1894,  52. 

35  Tamura,  Zeit.  f .  physiol.  Chemio,  Ixxxix,  1914,  304. 
™Iwanoff,  Hofmeister's  Beitr.,  1,  1902,  524. 
'"Fischer,  A.,  "Vorlesung  iiber  die  Bakt.,"  Jena,  1903. 


MORPHOLOGY,  REPRODUCTION,  ETC.  25 

form,  and  the  tliio  bacteria  contain  insoluble  sulphur  in  their  proto- 
plasm. 

The  chemical  constitution  of  bacteria  is  of  considerable  impor- 
tance in  connection  with  problems  of  immunization.  It  is  not  at  all 
sure  that  the  antigenic  substances  in  bacteria  consist  entirely  of 
proteins  of  the  albumin-globulin  variety.  Extracts  of  bacterial 
bodies,  such  as  those  we  have,  on  a  number  of  occasions,  produced 
with  typhoid  bacilli,  tubercle  bacilli,  etc.,  contain  very  large  amounts 
of  material,  non-coagulable  by  heat,  which  come  down  on  the  addi- 
tion of  weak  acetic  acid  in  the  cold,  and  which  are  soluble  only  in 
dilute  alkali,  such  as  the  antigenic  proteins  contained  in  many 
plants.  It  is  not  impossible  that  these  may  be  the  decisive  antigenic 
constituents,  rather  than  the  albumin- globulin  contents. 

Osmotic  Properties  of  the  Bacterial  Cell. — Like  all  other  animal 
and  vegetable  cells,  the  bacterial  cell  forms  in  itself  a  small  osmotic 
unit  which  reacts  delicately  to  differences  of  pressure  existing  be- 
tween its  own  protoplasm  and  the  surrounding  medium.  The  perfect 
and  normal  morphology  of  a  microorganism,  therefore,  can  exist 
only  when  the  osmotic  pressure  within  the  protoplasm  of  the  cell  is 
isotonic  or  equal  to  that  of  its  own  environment.  The  changes  pro- 
duced in  the  morphological  relations  of  a  cell  when  transferred  from 
one  environment  into  another  of  varying  osmotic  pressure,  depend 
intimately  upon  the  "permeability"  of  the  cell  membrane  for  dif- 
ferent substances.  When  such  a  membrane  is  permeable  for  water 
and  not  for  substances  in  solution,  it  is  technically  spoken  of  as 
"semi-permeable."  Now,  as  a  matter  of  fact,  the  bacterial  cell 
membrane  is  easily  permeable  for  water,  but  its  permeability  differs 
greatly  in  various  species  of  bacteria  for  other  substances.  Thus, 
for  instance,  the  cholera  vibrio  shows  great  permeability  for  common 
salt  and  B.  fluorescens  liquefaciens  shows  a  lower  permeability  for 
potassium  nitrate  than  do  many  other  bacteria.38 

When  a  microorganism  is  suddenly  removed  from  an  environ- 
ment of  low  osmotic  pressure  into  one  showing  a  high  pressure,  say, 
from  a  dilute  to  a  concentrated  solution  of  NaCl,  an  abstraction  of 
water  from  the  cell  occurs,  with  a  consequent  shrinkage  of  the  proto- 
plasm away  from  the  cell  membrane.  This  process  is  spoken  of  as 
"plasmolysis. "  Cell  death  does  not  usually  occur  with  plasmolysis, 
but  by  slow  diffusion  of  the  salt  itself  into  the  protoplasm,  the 


38  Gottschlich,  in  Fliigge,  ' '  Mikroorganismen, "  i,  p.  91. 


26  BIOLOGY  AND  TECHNIQUE 

equilibrium  may  eventually  be  restored  and  the  normal  morphology 
of  the  cell  resumed.  In  all  cases  the  speed  and  completeness  of  the 
return  to  normal  depends  upon  the  permeability  of  the  cell  mem- 
brane for  the  dissolved  substances.  There  is  no  evidence  to  support 
the  view  that  the  internal  pressure  of  a  cell  may  be  in  any  way 
increased  by  an  inherent  power  of  the  protoplasm  independently 
of  the  laws  of  diffusion.  As  a  general  rule,  old  cultures  are  more 
susceptible  to  plasmolysis  than  are  young  and  vigorous  strains. 
Spores  and,  according  to  A.  Fischer,39  flagella  are  much  less  sus- 
ceptible to  osmotic  changes  than  are  the  vegetative  bodies. 

When,  on  the  other  hand,  bacteria  are  suddenly  removed  from  a 
medium  possessing  a  high  osmotic  pressure  to  one  comparatively  low, 
say,  from  a  concentrated  salt  solution  to  distilled  water,  a  bursting 
of  the  cell  may  occur,  a  process  spoken  of  as  "plasmoptysis. "  Plas- 
moptysis  leads  to  cell  death,  and  is  probably  the  cause  of  the  death 
of  microorganisms  so  often  observed  in  distilled  water  emulsions  of 
bacteria. 

Other  Physical  Properties  of  Bacteria. — The  refractive  index  of 
the  vegetative  bacterial  body  is  low,  in  contrast  to  the  highly  refract- 
ive character  of  the  spores  and  flagella.  According  to  Fischer,  the 
ectoplasm  or  cell  membrane  shows  a  higher  index  than  does  the 
endoplasm. 

The  specific  gravity  of  various  microorganisms  has  been  investi- 
gated by  Bolt-on,40  Rubner,41  and  others.  Some  of  Rubner's  results 
are  the  following: 

Gelatin  fluidifiers   Sp.  gr.  1.0651 

Gas  formers   "     "  1.0465 

Cultures  from  potato ' '      ' '  1.038 

M.  prodigiosus "      ' '  1.054 


89  A.  Fischer,  quoted  from  Gottschlich  in  Fliigge,  "Mikroorganismen, "  I,  p.  91. 

40  Bolton,  Zeit.  f .  Hyg.,  i,  1886. 

41  Rubner,  Arch.  f.  Hyg.,  xi,  1890. 


CHAPTER  III 

THE   RELATION   OF   BACTERIA   TO   ENVIRONMENT,    AND    THEIR 
CLASSIFICATION 

NUTRITION  OF  BACTERIA 

LIKE  all  protoplasmic  bodies,  bacteria  consist  of  carbon,  oxygen, 
hydrogen,  and  nitrogen,  to  which  are  added  inorganic  salts  and  vary- 
ing quantities  of  phosphorus  and  sulphur.  In  order  that  bacteria 
may  develop  and  multiply,  therefore,  they  must  be  supplied  with 
these  substances  in  proper  quantity  and  in  forms  suitable  for  assimi- 
lation. To  formulate  definite  laws  based  on  chemical  structure  as  to 
the  compounds  suitable,  and  those  unsuitable  for  use  by  the  bacteria, 
is  obviously  impossible  owing  to  the  great  metabolic  variations  exist- 
ing within  the  bacterial  kingdom,  and  notable  attempts  to  do  so, 
such  as  those  by  Loew,1  have  not  successfully  withstood  critical 
inquiry.  So  unlike  are  the  food  requirements  of  bacteria  that  a  basis 
for  identification  even  among  closely  related  species  may  often  be 
found  in  differences  in  metabolism  of  carbohydrates,  and  the  sugges- 
tion has  frequently  been  made  that  with  further  knowledge  of  food 
requirements  would  come  a  more  logical  classification  of  microor- 
ganisms than  any  at  present  available.2 

Carbon. — The  carbon  necessary  for  bacterial  nourishment  or  anab- 
olism  may  be  obtained  either  directly  from  proteins,  carbohydrates, 
and  fats,  or  from  the  simpler  derivatives  of  these  substances.  Thus, 
the  amino-acids,  ketons,  and  organic  acids,  like  tartaric,  citric,  and 
acetic  acids,  glycerin,  and  even  some  of  the  alcohols,  may  furnish 
carbon  in  a  form  suitable  for  bacterial  assimilation.  A  limited  num- 
ber of  bacterial  species,  furthermore,  notably  the  nitrobacteria  of 
Winogradsky,  are  capable  of  obtaining  their  required  carbon  from 
atmospheric  C02,  and  possibly  from  other  simple  carbon  compounds 
added  to  culture  media.3 

1  Loew,  Cent,  f .  Bakt.,  I,  xii,  1892. 
-Doryland,  Jour,  of  Bacter.,  1,  1916,  135. 
'Hunts,  Compt.  rend,  de  Tacad.  des  sciences,  t.  iii. 

27 


28  BIOLOGY  A 

Oxygen. — Oxygen  is  obtained,  by  the  large  majority  of  bacteria, 
directly  from  the  atmosphere  in  the  form  of  free  O2.  For  many 
microorganisms,  moreover,  the  presence  of  free  oxygen  is  a  necessary 
condition  for  growth.  These  are  spoken  of  as  the  "obligatory 
aerobes."  Among  the  pathogenic  bacteria  proper,  many,  like  the 
gonococcus,  bacillus  influenza*,  and  bacillus  pestis,  show  a  marked 
preference  for  a  well-oxygenated  environment.  Probably  there  is 
no  pathogenic  microorganism  which,  under  certain  conditions  of 
nutrition,  is  entirely  unable  to  exist  and  multiply  in  the  complete 
absence  of  this  gas.  The  conditions  existing  within  the  infected 
animal  organism  cause  it  to  seem  likely  that  all  incitants  of  infec- 
tion may,  at  times,  thrive  in  the  complete  absence  of  free  oxygen. 

There  is  another  class  of  organisms,  on  the  other  hand,  for  whose 
development  the  presence  of  free  oxygen  is  directly  injurious.  These 
microorganisms,  known  as  "obligatory  anaerobes,"  obtain  their 
supply  of  oxygen  indirectly,  by  enzymatic  processes  of  ferments 
and  proteolytic  cleavage,  from  carbohydrates  and  proteins,  or  by 
reduction  from  reducible  bodies.  Among  the  pathogenic  microor- 
ganisms the  class  of  "obligatory  anaerobes"  is  represented  chiefly 
by  Bacillus  tetani,  the  bacillus  of  malignant  edema,  the  bacillus  of 
symptomatic  anthrax.  Bacillus  aerogenes  capsulatus.  and  Bacillus 
botulinus. 

Intermediate  between  these  two  classes  is  a  large  group  of  bac- 
teria which  thrive  well,  both  under  aerobic  and  anaerobic  conditions. 
Some  of  these,  which  have  a  preference  for  fre-  _  D  but  never- 
theless possess  the  power  of  thriving  under  anaerobic  conditions,  are 
spoken  of  as  "facultative  anaerobes."  In  others  the  reverse  of  this 
is  true ;  these  are  spoken  of  as  "facultative  aerobes."  These  vari- 
of  bacteria  are  by  far  the  most  numerous  and  comprise  most  of  our 
parasitic  and  saprophytic  bacteria. 

The  relation  of  microorganisms  to  oxygen  is  extremely  subtle, 
therefore,  and  not  to  be  biologically  dismissed  by  a  rigid  classifica- 
tion into  aerobes,  facultative  anaerobes,  and  obligatory  anaerobes. 
Both  Engelmaiin,4  by  a  method  of  observing  motile  bacteria  in  the 
hanging  drop  as  to  their  behavior  in  relation  to  the  oxygen  given 
off  by  a  chlorophyll-bearing  alga,  and  Beijerinck  macroscopic 

method  of  observing  similar  bacteria  as  to  their  motion  away  from 

*  Er,,-jflmanfir  Botanisohe  Zeitung,  1881. 
nt.  f.  Bakt.,  I 


RELATION   TO   ENMRONMENT— CLASSIFICATION  29 

or  toward  an  oxygenated  area,  were  able  to  demonstrate  delicately 
graded  variations  between  species,  favoring  various  degrees  of 
oxygen  pressure. 

The  discovery  by  Pasteur  that  certain  bacteria  develop  only  in 
the  absence  of  free  oxygen,  produced  a  revolution  in  our  conceptions 
of  metabolic  processes,  since  up  to  that  time  it  was  believed  that  life 
could  be  supported  only  when  a  free  supply  of  O2  was  obtainable. 

reur's  original  explanation  for  this  phenomenon  was  that  anae- 
robic conditions  of  life  were  always  associated  with  some  form  of 
carbohydrate  fermentation  and  that  oxygen  was  obtained  by  these 
microorganisms  by  a  splitting  of  carbohydrates.  As  a  matter  of 
fact,  for  a  large  number  of  microorganisms,  this  is  actually  true,  and 
the  presence  of  readily  fermentable  carbohydrates  not  only  increases 
the  growth  energy  of  a  large  number  of  anaerobic  bacteria,  but  in 
many  cases  permits  otherwise  purely  aerobic  bacteria  to  thrive  under 
anaerobic  conditions.6  On  the  other  hand,  the  basis  of  anaerobic 
growth  can  not  always  be  found  in  the  fermentation  of  carbo- 
hydrates or  in  the  simple  process  of  reduction.  A  number  of  strictly 
anaerobic  bacteria  may  develop  in  the  entire  absence  of  carbohy- 
drates or  reducing  substances,  obtaining  their  oxygen  supply  from 
other  suitable  sources,  some  of  which  may  be  the  complex  proteins. 
Thus  the  tetanus  bacillus  may 7  thrive  when  the  nutritive  substances 
in  the  media  are  entirely  protein  in  nature. 

The  favorable  influence  of  certain  actively  reducing  bodies,  like 
sodium  formate  or  sodium-indigo-sulphate,  upon  anaerobic  cultiva- 
tion is  probably  referable  to  their  ability  to  remove  free  oxygen  from 
the  media  and  thus  perfect  the  anaerobiosis.8 

Til  like  plants  which  derive  much  of  their  energy  for  growth 
from  the  sun's  rays,  bacteria,  with  the  exception  of  the  pigmented 
sulphur  bacteria,  are  dependent  upon  their  food  supply  as  a  source 
of  energy.  To  some  extent  a  simple  oxidation  of  carbohydrates  and 
other  substances  takes  place  by  means  of  atmospheric  oxygen  with 
the  production  of  CO2.  There  is  thus  a  type  of  true  respiration  in 
bacteria  with  the  absorption  of  oxygen  and  elimination  of  CO2.  Not 
all  the  absorbed  oxygen  reappears  as  CO2  and  most  of  the  remainder 
is  probably  used  up  in  the  formation  of  water  or  goes  into  the  struc- 
ture of  the  bacterial  cell.  The  production  of  CO2  has  also  been 

•  Theobald  Smith,  Cent.  f.  Bakt.,  I,  xviii,  1895. 
1  Chudiokow,  Cent,  f .  Bakt.,  Kef.,  II,  iv,  1898. 
»  KUasato  and  Weyl,  Zeit.  f.  Hyg.,  viii,  1890. 


30  BIOLOGY  AND  TECHNIQUE 

shown  to  occur  in  anaerobic  cultures  where  the  oxygen  must  have 
come  from  some  constituent  of  the  medium.  It  is  probable  that 
bacterial  respiration  is  frequently  of  the  anaerobic  type  in  which, 
for  example,  such  compounds  as  nitrates  are  reduced  to  nitrites  and 
free  nitrogen.  Energy  may  be  produced,  however,  by  the  oxidation 
of  other  elements  than  carbon  and  hydrogen;  thus,  certain  of  the 
iron  bacteria  seem  to  obtain  energy  from  the  oxidation  of  ferrous 
to  ferric  compounds  in  their  protoplasm,  and  the  sulphur  bacteria 
oxidize  H2S  to  free  sulphur.  The  colored  sulphur  bacteria  carry  out 
this  change  under  the  combined  influence  of  sunlight  and  their  pig- 
ment in  this  way  resembling  the  green  plants,  but  the  unpigmented 
forms  do  not  require  radiant  energy. 

While  processes  of  oxidation  are  perhaps  the  most  important 
source  of  energy  for  certain  bacteria,  for  other  types  and  under 
different  conditions  chemical  reactions  of  another  kind  serve  to  pro- 
duce energy.  Processes  of  fermentation  and  perhaps  of  decay  and 
putrefaction  liberate  considerable  quantities  of  energy.  For  ex- 
ample, alcoholic  fermentation  in  which  neither  oxidation  nor  reduc- 
tion is  involved  produces  about  33  calories  for  each  gram  molecule 
of  sugar  fermented.  Since  complete  oxidation  of  the  same  quantity 
of  sugar  results  in  the  liberation  of  about  680  calories  it  is  apparent 
that  relatively  large  amounts  of  glucose  must  be  fermented  to  supply 
microorganisms  with  sufficient  energy  for  their  growth  and,  as  a 
matter  of  fact,  bacteria  will  ferment  many  times  their  weight  of 
carbohydrates.  Acid  fermentation,  such  as  the  lactic  fermentation 
of  milk  and  probably  many  of  the  processes  of  protein  cleavage,  are 
exothermic  and  will  supply  bacteria  with  energy.  Obviously  these 
reactions  are  particularly  adapted  to  anaerobic  growth  since  for 
many  of  them  oxygen  is  not  required. 

While  a  profuse  supply  of  oxygen  absolutely  inhibits  the  growth 
of  most  anaerobes,  a  number  of  these  may,  nevertheless,  develop 
when  only  small  quantities  of  oxygen  are  present.  Minute  quantities 
of  free  oxygen  in  culture  media  have  been  shown  by  Beijerinck9 
and  others  not  to  inhibit  the  growth  of  Bacillus  tetani  and  Theobald 
Smith10  has  recently  demonstrated  that  when  suitable  nutritive 
material  in  the  form  of  fresh  liver  tissue  is  added  to  bouillon,  a  num- 
ber of  anaerobic  bacteria  may  be  induced  to  grow  in  indifferently 


9  Beijerinck,  Cent.  f.  Bakt.,  II,  vi,  1900. 

10  Tli.  Smith,  Brown,  and  Walker,  Jour.  Mecl.  Res.,  ix,  1906. 


RELATION  TO  ENVIRONMENT— CLASSIFICATION  31 

anaerobic  environment.  Fe'rran11  succeeded  in  adapting  the  tetanus 
bacillus  to  an  aerobic  environment.  In  this  case,  however,  the  viru- 
lence of  the  bacillus  was  lost. 

Nitrogen. — The  nitrogen  required  by  bacteria  is  taken,  in  most 
cases,  from  proteins,  and  many  of  the  non-diffusible  albumins  may  be 
rendered  assimilable  by  the  proteolyzing  enzymes  possessed  by  micro- 
organisms. Among  the  pathogenic,  more  strictly  parasitic  bacteria, 
moreover,  a  delicate  specialization  may  be  observed  as  to  the  partic- 
ular varieties  of  animal  albumin  which  may  be  utilized  by  them. 
Thus  the  gonococcus  grows  more  readily  only  upon  uncoagulated 
human  blood  serum,  and  the  diphtheria  bacillus  outgrows  other  bac- 
teria upon  a  medium  composed  for  the  greater  part  of  coagulated 
beef  serum.  For  bacteria  that  do  not  require  native  animal  protein 
for  their  development,  the  most  common  nitrogenous  ingredient  of 
culture  media  is  peptone. 

Many  bacteria  (pathogenic  and  saprophytic),  on  the  other  hand, 
may  thrive  on  media  containing  absolutely  no  protein,  in  which  case, 
of  course,  a  synthesis  of  protein  must  be  assumed.  A  medium  used 
to  demonstrate  this,  devised  by  Uschinski,12  contains  ammonium  lac- 
tate,  glycerin,  asparagin  (the  amine  of  anino-succinic  acid),  and 
inorganic  salts. 

Creatin,  creatinin,  urea  and  urates,  and  even  ammonia  compounds 
and  nitrates,  may  serve  as  sources  of  nitrogen  for  many  of  the  less 
parasitic  bacteria.  A  limited  number  of  species,  moreover,  the 
bacilli  in  the  root  tubercles  of  the  leguminosae  and  the  nitrogen- 
fixing  organisms  of  the  soil,  can  obtain  their  nitrogen  directly  from 
the  free  N2  of  the  atmosphere. 

Although  the  sources  of  carbonaceous  and  of  nitrogenous  food 
have  been  separately  discussed,  it  should  not  be  forgotten  that,  in 
many  instances,  both  elements  are  taken  up  within  the  same  com- 
pound, and  that  separate  supplies  are  a  necessity  in  isolated  cases 
only. 

Hydrogen. — Hydrogen  is  obtained  by  bacteria  largely  in  combi- 
nation as  water  and  together  with  the  carbon  and  nitrogen  contain- 
ing substances. 

Salts. — The  phosphatic  constituents  of  the  bacterial  body  are 
taken  in,  chiefly,  as  phosphates  of  magnesium,  calcium,  sodium,  or 


11  Ferran,  Cent,  f .  Bakt.,  I,  xxiv,  1898. 

12  UschinsTci,  Cent,  f .  Bakt.,  I,  xiv,  1893. 


32  BIOLOGY  AND  TECHNIQUE 

potassium.  The  phosphates  seem  to  be  necessary  constituents  of  cul- 
ture media,  while  chlorides,  on  the  other  hand,  according  to  Pros- 
kauer13  and  Beck,  are  not  absolutely  essential.  Sodium  salts,  as  a 
rule,  seem  to  be  more  advantageous  for  purposes  of  bacterial  culti- 
vation than  potassium  salts. 

The  uncombined  sulphur,  which  is  often  a  constituent  of  bacteria, 
is  usually  supplied  by  soluble  sulphates.  In  the  case  of  the  thio- 
bacteria  of  Winogradsky,  however,  free  H2S  is  necessary  for  its 
formation.14 

The  relative  quantities  of  various  nutrients  in  culture  media  are 
important  in  so  far  as  too  high  concentrations  may  inhibit  growth. 
In  this  respect,  however,  separate  species  vary.  The  development  of 
bacteria  is  far  oftener  arrested  by  the  accumulation  of  waste  prod- 
ucts than  by  an  exhaustion  of  nutrient  materials. 

Substances  of  Unknown  Composition. — Although  the  necessity 
for  supplying  bacteria  with  all  the  elements  of  protoplasm  is  obvi- 
ous, many  forms,  particularly  among  the  pathogenic  bacteria,  will 
fail  to  grow  on  media  composed  of  amino-acids  or  peptones  together 
with  carbohydrates  and  mineral  material.  So  far  as  is  known,  no 
chemical  element  necessary  for  growth  is  lacking  in  such  a  medium, 
and  the  failure  of  bacteria  to  multiply  must  indicate  either  that  car- 
bon and  nitrogen  are  not  present  in  suitable  combination,  or  that 
some  special  accessory  chemical  grouping  is  wanting.  For  many 
practical  purposes  such  media  may  be  rendered  suitable  by  the  addi- 
tion of  meat  extract  or  meat  infusion.  The  components  of  meat  in- 
fusion which  the  bacteria  utilize  in  their  growth  have  not  been 
isolated.  They  seem  to  be  somewhat  labile,  chemically,  and  the 
infusion  loses  its  power  to  promote  growth  under  relatively  simple 
chemical  manipulation.  That  there  is  more  than  one  necessary  com- 
ponent is  indicated  by  recent  work  15  in  which  it  has  been  shown 
that  boiling  the  infusion  with  charcoal  renders  it  unsuitable  for  the 
growth  of  certain  bacteria,  while  the  addition  of  an  acid  hydrolysate 
of  casein  and  of  some  other  proteins  renders  it  again  satisfactory, 
although  the  reactivating  substance  is  probably  not  one  of  the  known 
amino  acids,  and  will  not  itself  produce  growth  of  the  bacteria  with- 
out the  decolorized  meat  infusion. 


13  Proslcauer  and  Beck,  Zeit.  f .  Hyg.,  xviii,  1895. 

14  Voges,  Cent.  f.  Bakt.,  I,  xviii,  1893. 

15  Mueller,  Proc.  Soc.  Exp.  Bi6l.  and  Med.,  1920-21. 


RELATION  TO  ENVIRONMENT— CLASSIFICATION  33 

Meat  infusion,  however,  will  not  suffice  for  some  of  the  more 
strictly  parasitic  species,  such  as  the  meningococcus  and  gonococcus, 
and  for  satisfactory  growth  of  these  organisms  a  highly  complex 
substance,  such  as  whole  blood  or  serum,  is  necessary.  It  is  not  cer- 
tain at  present  whether  the  required  material  is  the  whole  protein 
or  some  simpler  substance  occurring  in  the  animal  body.  For  ex-  • 
ample,  the  influenza  bacillus  has  been  known  for  a  long  time  to 
require  red  blood  cells  for  its  successful  cultivation.  It  was  formerly 
believed  that  the  hemaglobin  was  the  essential  factor  required  by 
this  organism,  but  recently  it  has  been  shown16  that  if  media  are 
prepared  by  the  addition  of  blood  followed  by  heatirig  to  70°  to  75°, 
and  filtering  the  coagulum,  much  better  growth  is  obtained  than  on 
ordinary  blood  agar.  Since  the  proteins,  including  the  hemaglobin, 
have  been  removed  by  coagulation  and  filtration,  probably  some  non- 
protein  component  of  the  red  cell  is  required  by  this  organism. 

Lloyd 17  has  suggested  that  these  substances  present  in  blood  tai\d 
animal  fluids  resemble  the  vitamines  which  are  now  recognized  as 
playing  such  an  important  role  in  animal  metabolism.  She  found  that 
they  were  often  removed  from  solution  by  filtering  through  paper 
or  cotton,  and  on  this  observation  are  based  the  extremely  useful 
hormone  or  vitamine  media  of  Huntoon. 18  Whether  or  not  these 
substances  are  identical  with,  or  merely  resemble,  the  vitamines  of 
animal  foods  remains  to  be  established.  It  has  been  recently  shown; 
however,  that  ordinary  yeast 19  and  Sclerotinia  cinerea  the  organ- 
ism,20 which  causes  brown  rot  disease  in  fruits,  require  water  soluble 
B.  vitamine  for  their  growth. 

PARASITISM  AND  SAPROPHYTISM 

When  we  speak  of  bacteria  as  parasites  or  as  saprophytes,  we 
classify  them,  primarily,  according  to  their  relationship  to  the  bodies 
of  higher  animals.  "Parasites"  are  those  bacteria  which  are  capable 
of  living  and  multiplying  within  the  human  or  animal  body,  whereas 
the  term  "  saprophytes "  refers  to  the  multitude  of  microorganisms 


16  Fleming,  Lancet,  1919,  I,  138. 

17  Lloyd,  J.  Path,  and  Bact.,  21,  1916,  113. 

18  Huntoon,  J.  Inf.  Dis.,  23,  1918,  169. 

19  Williams,  J.  Biol.  Chem.,  38,  1919,  465. 

20  Willaman,  J.  Am.  Chem.  Soc.,  42,  1920,  549. 


34  BIOLOGY  AND  TECHNIQUE 

which  are  unable  to  hold  their  own  under  the  environmental  condi- 
tions found  in  the  tissues  of  higher  animals,  but  are  found,  almost 
ubiquitously,  in  air,  soil,  manure,  and  water.  The  separation  is  by  no 
means  a  sharp  one  and  carries  with  it  other  implications,  which  the 
use  of  these  terms  always  conveys.  While  parasites  are  usually  very 
fastidious  as  to  nutritional  and  temperature  requirements,  most 
saprophytes  are  easily  cultivated  upon  the  simplest  media.  Thus 
certain  parasitic  bacteria,  such  as  the  bacillus  of  influenza,  the  gono- 
coccus,  and  others,  are  dependent  upon  specific  forms  of  animal 
proteins  for  their  food  supply,  while  typical  saprophytes,  like  Bacil- 
lus proteus,  may  thrive  and  multiply  upon  even  the  simplest  organic 
protein  derivatives. 

Between  the  strict  parasites  and  the  saprophytes,  there  is  a  large 
class  of  bacteria,  to  which  the  majority  of  pathogenic  varieties 
belong,  the  members  of  which  are  capable  of  developing  luxuriantly 
under  both  conditions.  These  bacteria  are  often  spoken  of  as  facul- 
tative parasites. 

More  recently  the  question  of  parasitism  and  saprophytism  has 
become  closely  interwoven  with  our  conceptions  of  virulence.  Bail 
(see  section  on  Aggressins)  has  classified  parasites  into  strict  para- 
sites and  half  parasites.  By  the  first  term  he  designates  bacteria  like 
Bacillus  anthracis,  which  actually  invade  all  the  tissues  of  their  host, 
while,  by  the  term  "half  parasites,"  he  refers  to  microorganisms  like 
the  spirillum  of  cholera  which  gain  a  foothold  upon  some  part  of  the 
body  of  the  host,  but  do  not  actually  penetrate  into  the  general  cir- 
culation. 

All  pathogenic  bacteria,  therefore,  must  be  grouped  as  parasites, 
strict  or  facultative,  while  the  saprophytes,  as  a  class,  perform  the 
far  more  important  task  of  breaking  up  organic  matter  outside  of 
the  animal  body,  by  putrefaction  and  fermentation.  Absolute  sepa- 
ration between  the  two  classes,  however,  can  not  be  maintained,  since 
many  ordinarily  saprophytic  bacteria  may  display  parasitic  qualities 
if  administered  in  large  numbers  to  animals  or  man  in  whom  resist- 
ance to  bacterial  invasion  is  at  a  low  ebb. 

ANTAGONISM  AND  SYMBIOSIS  OF  BACTERIA 

The  ubiquity  of  bacteria  in  nature  of  course  implies  the  simulta- 
neous presence  of  many  species  in  all  places  where  special  conditions 
have  provided  a  favorable  environment  for  growth.  Thus  bacterio- 


RELATION   TO   ENVIRONMENT— CLASSIFICATION  35 

logical  investigation  of  water,  milk,  manure,  soil,  or  organic  infu- 
sions always  reveals  the  presence  of  a  large  number  of  different 
varieties  within  the  same  substance.  If  the  food  supply  in  such  a 
natural  culture  is  at  all  limited  in  quantity,  or  the  removal  of  waste 
products  is  prohibited,  it  will  usually  be  found  that  gradually  the 
numbers  of  varieties  will  diminish  and  a  few  species,  or  even  only 
one,  will  prevail.  In  the  case  of  milk,  for  instance,  after  standing 
for  three  or  four  days  at  a  suitable  temperature,  two  or  three 
varieties  will  be  found  to  have  taken  the  place  of  the  twenty  or 
thirty,  which  may  have  been  present  originally. 

This  behavior  is  due  to  the  influence  which  various  microorgan- 
isms exert  upon  each  other  and  is  known  as  antagonism.  Such  antag- 
onism probably  depends  upon  the  fact  that  the  metabolic  products  of 
the  predominant  species  (the  one  or  ones  for  whom  the  special  cul- 
tural conditions  are  most  favorable)  inhibit  the  growth  of  the  less 
vigorous  varieties.  Many  examples,  experimentally  supported,  of 
such  antagonism,  can  be  given.  Thus,  the  gonococcus  is  distinctly 
inhibited  by  the  soluble  products  of  Bacillus  pyocyaneus,21  while  in 
the  preesnce  of  pyogenic  cocci  it  develops  luxuriously,  and  the  bacil- 
lus of  plague  is  completely  inhibited  when  streptococci  are  present 
in  the  culture.22 

Mutual  inhibition  may  also  be  due  to  the  monopolizing  of  the 
nutrition  in  the  medium  by  the  predominating  species  or  to  the 
change  in  reaction  produced  by  its  growth.  This  last  consideration 
is  probably  the  secret  of  the  inhibitory  effect  exerted  by  acid-pro- 
ducers upon  bacteria  of  putrefaction,  and  has  received  practical  the- 
rapeutic application  in  MetchnikofPs  lactic-acid  bacillus  therapy, 
described  elsewhere. 

When  simultaneous  presence  of  two  species  in  the  same  environ- 
ment favors  the  development  of  both,  the  condition  is  spoken  of  as 
symbiosis.  Such  dependence  is  not  so  frequent  as  antagonism,  but  it 
does  occur.  Examples  of  such  a  condition  have  been  observed  in 
cultures  containing  diphtheria  bacilli  and  streptococci23  and  have 
been  frequently  observed  in  cultures  containing  both  aerobic  and 
anaerobic  bacteria,  where  the  former  favor  the  development  of  the 
latter  by  monopolizing  the  supply  of  free  oxygen.  Symbiosis  may 


21  Schafcr,  Fortschr.  d.  Med.,  5,  1896. 

22  Bitter,  Rep.  Egypt  Plague  Com.,  Cairo,  1897. 

23  Hilbert,  Zeit.  f .  Hyg.,  xxix,  1895. 


36  BIOLOGY  AND  TECHNIQUE 

also  take  place  in  cultures  in  which  complex  food  products  are  split 
up  by  one  species,  furnishing  substances  for  ingestion  by  species  with 
a  lesser  digestive  ability. 

RELATIONS  OF  BACTERIA  TO  PHYSICAL  ENVIRONMENT 

Relation  of  Temperature. — Like  all  other  living  beings,  bacteria 
develop  and  multiply  by  virtue  of  a  series  of  chemical  and  physical 
processes,  by  means  of  which  growth  energy  is  obtained  by  destruc- 
tion or  catabolism,  and  the  lost  tissues  resupplied  by  absorption  of 
nutritive  materials.  It  is  natural,  therefore,  that  the  conditions  of 
external  temperature  should  intimately  affect  the  metabolic  proc- 
esses. The  range  of  temperature  at  which  bacteria  may  grow  is  sub- 
ject to  wide  variations  among  different  species.  Each  species,  on  the 
other  hand,  may  thrive  within  a  more  or  less  elastic  range  of  tem- 
perature, each  one  having  an  optimum,  a  minimum,  and  a  definite 
maximum  temperature.  When  the  optimum  temperature  is  present 
in  the  environment,  the  functions  of  absorption  and  excretion  keep 
pace  with  each  other,  and  the  chemical  balance  is  well  preserved. 
When  the  temperature  is  lower  than  the  optimum,  all  metabolic 
processes  take  place  more  slowly,  and  the  bacterium  gradually  enters 
into  a  resting  or  latent  stage,  at  which  actual  growth  may  be  ex- 
ceedingly slow  or  entirely  inhibited.  When  the  temperature  is  higher 
than  the  optimum,  the  destructive  processes  are  carried  on  more 
rapidly  than  the  substitution  of  waste  products  by  absorption,  and  a 
gradual  weakening  of  vital  energy,  or  even  a  gradual  death  of  the 
bacterium,  may  take  place.  When  certain  bacteria  form  spores,  they 
become  very  much  more  resistant  against  both  high  and  low  temper- 
atures, probably  because  a  true  resting  stage  has  been  reached,  dur- 
ing which  metabolism  has  been  reduced  to  a  minimum,  there  being 
practically  no  nutritive  material  taken  in  and  correspondingly  little 
destruction  taking  place  within  the  body  of  the  microorganism. 

The  optimum  temperature  for  various  bacteria  depends  upon  the 
habitual  environment,  in  which  the  particular  species  is  accustomed 
to  exist.  Thus,  for  the  large  majority  of  bacteria  pathogenic  for 
human  beings,  the  optimum  temperature  is  at  or  about  37.5°  C. 
There  are  a  large  number  of  bacteria  common  in  water,  however, 
which  grow  hardly  at  all  at  the  body  temperature,  but  thrive  most 
luxuriantly  at  temperatures  of  about  20°  C.  F.  Forster,24  moreover, 


24  F.  Forster,  Cent,  f .  Bakt.,  ii,  1887. 


RELATION   TO  ENVIRONMENT— CLASSIFICATION  37 

described  certain  phosphorescent  bacteria,  isolated  from  sea-water, 
which  grow  readily  at  0°  C.,  or  a  little  above.  On  the  other  hand, 
Miquel 25  has  described  non-motile  bacilli,  which  he  isolated  from  the 
water  of  the  Seine,  which  grew  rapidly  at  temperatures  ranging 
apout  70°  C.,  and  the  so-called  "mucedinees  thermophiles, "  de- 
scribed by  Tsiklinski,26  develop  most  readily  at  temperatures  very 
little  above  this.  It  is  thus  plain  that  the  temperatures  favored  by 
various  bacteria  depend  to  a  large  extent  upon  an  adaption  of  these 
bacteria  through  many  generations  to  specific  environmental  condi- 
tions. A  good  illustration  of  this  is  furnished  by  the  bacillus  of 
avian  tuberculosis,  a  microorganism  differing  essentially  from  the 
bacillus  of  human  tuberculosis  in  that  its  optimum  growth  tempera- 
ture lies  at  41°-42°  C.,  a  temperature  which  exceeds  the  optimum 
temperature  for  the  human  type  by  as  much  as  the  normal  tempera- 
ture of  birds  exceeds  that  of  man.  The  same  principle  is  illustrated 
by  the  facts  that  the  bacteria  which  have  a  very  low  optimum  tem- 
perature are  usually  those  isolated  from  water,  and  the  so-called 
thermophile  or  high-temperature  bacteria  are  obtained  from  hot 
springs  and  from  the  upper  layers  of  the  soil,  where,  according  to 
Globig,27  occasionally  temperatures  ranging  from  about  55°  C.  occur. 

As  stated  before,  one  and  the  same  species  may  develop  within  a 
wide  temperature  range,  and  it  may  be  possible,  by  persistent  culti- 
vation at  special  temperatures,  to  adapt  certain  bacteria  to  grow  lux- 
uriantly at  temperatures  removed  by  several  degrees  from  their 
normal  optimum.  In  such  cases  it  may  often  occur  that  special 
characteristics  of  the  given  species  may  be  lost.  An  example  of  this 
is  the  loss  of  virulence  and  of  spore-formation  which  takes  place 
when  anthrax  bacilli  are  cultivated  at  42°  C.,  or  the  loss  of  the  power 
to  produce  pigment  when  bacillus  prodigiosus  is  grown  at  tempera- 
tures above  30°  C. 

The  vegetative  forms  of  most  of  the  pathogenic  bacteria  may 
grow  at  temperatures  ranging  between  20°  and  40°  C.  This  can, 
however,  by  no  means  be  regarded  as  applicable  to  all  of  the  patho- 
genic bacteria,  as  some  of  these,  like  the  gonococcus,  the  pneumo- 
coccus,  the  tubercle  bacillus,  and  others,  are  delicately  susceptible  to 
temperature  changes  and  have  the  power  of  growing  only  within 


25  Miquel,  Bull,  de  la  Stat.  Munic.  de  Paris,  1879. 
28  TsiTdinski,  Ann.  Past.,  1889. 
27  Globig,  Zeit.  f.  Hyg.,  iii. 


38  BIOLOGY  AND  TECHNIQUE 

limits  varying  but  a  few  degrees  from  their  optimum.  Others,  on 
the  other  hand,  like  bacilli  of  the  colon  group,  Bacillus  anthracis, 
Spirillum  choleras  asiaticae,  etc.,  may  develop  at  temperatures  as  low 
as  10°  C.  and  as  high  as  40°  C.,  or  over.  The  range  of  temperature 
at  which  saprophytic  bacteria  may  develop  is  usually  a  far  wider 
one.  When  temperatures  exceed  in  any  considerable  degree  the 
maximum  growth  temperature,  the  vegetative  forms  of  bacteria 
perish.  Thus,  ten  minutes'  exposure  to  a  temperature  of  between 
55°  and  60°  C.  causes  death  of  the  vegetative  forms  of  most  micro- 
organisms. Death  in  such  cases  is  due  probably  to  a  coagulation  of 
the  protoplasm,  and  since  all  such  processes  of  coagulation  take  place 
best  in  the  presence  of  water,  the  thermal  death  point  of  most  bac- 
teria is  lower  when  heat  is  applied  in  the  form  of  boiling  water  or 
steam,  than  when  employed  as  dry  heat.  (See  section  on  Steriliza- 
tion.) 

When  spores  are  present  in  cultures,  the  resistance  to  heat  is  enor- 
mously increased.  Exactly  what  the  explanation  of  this  is  can  not 
at  present  be  stated.  It  may  be  that  the  high  concentration  in  which 
the  protoplasmic  mass  is  found  in  the  spores  renders  it  less  easily 
coagulable  than  is  the  protoplasm  of  the  vegetative  body.  A  more 
detailed  discussion  of  these  relations  will  be  found  in  the  section  on 
Heat  sterilization. 

The  thermal  death  points  of  many  bacteria  have  been  carefully 
studied  by  Sternberg,28  by  a  technique  described  elsewhere. 

The  thermal  death  points  ascertained  by  him  in  this  way,  with  an 
exposure  of  ten  minutes  in  a  fluid  medium,  for  some  of  the  more 
common  non-sporogenic  bacteria  are  as  follows : 

Spirillum  choleras  asiaticse 52°  C. 

Diplococcus  pneumonise   52°  C. 

Streptococcus  pyogenes    54°  C. 

Bacillus  typhosus    56°  C. 

Bacillus  pyocyaneus   56°  C. 

Bacillus  mucosus  capsulatus 56°  C. 

Bacillus  prodigiosus   58°  C. 

Staphylococcus  pyogenes  aureus 58°  C. 

Gonococcus    60°  C. 

Staphylococcus  pyogenes  albus 62°  C. 

The  bacillus  tuberculosis,  though  not  a  spore  bearer,  seems  to  be 
slightly  more  resistant  to  heat  than  other  purely  vegetative  microor- 

28  Sternberg,  "Textbook  of  Bacteriology,"  New  York,  1901. 


RELATION   TO  ENVIRONMENT— CLASSIFICATION  39 

ganisms.  Thus,  according  to  Smith29  and  others,  ten  and  twenty 
minutes'  exposure  to  a  temperature  of  70°  C.  is  necessary  to  destroy 
tubercle  bacilli  in  a  fluid  medium.  For  the  effectual  destruction  of 
spores  by  moist  heat,  a  temperature  of  100°  C.,  or  boiling  point,  is 
usually  necessary. 

Low  temperatures  are  much  less  destructive  than  the  high  ones, 
and  are  even  in  a  number  of  cases  useful  in  keeping  bacteria  alive  for 
long  periods,  inasmuch  as  metabolic  processes  are  inhibited  and  life 
is  maintained  without  actual  development  in  a  sort  of  resting  state. 
Actual  destruction  by  low  temperatures  rarely  takes  place.  The 
exposure  of  diphtheria,  typhoid,  and  other  bacilli  to  temperatures  as 
low  as  200°  C.  below  zero  has  been  carried  out  without  destruction 
of  the  microorganisms,  a  fact  which  is  of  great  importance  in  consid- 
ering the  possibility  of  infection  by  the  vehicle  of  ice.  Meningococci 
and  gonococci,  on  the  other  hand,  die  out  rapidly  when  exposed 
to  0°  C. 

Relation  to  Pressure. — High  pressure  does  not  injure  bacteria. 
Certes 30  found  that  a  pressure  of  two  atmospheres  had  no  influence 
upon  the  growth  of  anthrax  bacilli  suspended  in  blood. 

Relation  to  Moisture. — For  the  growth  and  development  of  all 
bacteria,  the  presence  of  water  is  necessary.  Nutritive  materials  can 
not  be  absorbed  by  an  osmotic  process  unless  in  a  state  of  solution. 
While  complete  dryness  does  not  permit  growth,  its  destructive 
action  upon  various  bacteria  is  subject  to  great  differences.  The 
effect  of  complete  drying  upon  bacteria  will  be  found  more  fully  dis- 
cussed on  page  76.  In  the  same  place  may  be  found  a  discussion  of 
the  effects  of  light,  electricity,  x-ray,  and  radium  rays  upon  bacteria. 

THE  CLASSIFICATION  OF  BACTERIA 

Too  simple  in  structure,  too  varied  in  biological  properties  to  be 
definitely  identified  with  either  the  vegetable  or  animal  kingdom,  the 
bacteria  are  placed  at  the  bottom  of  the  scale  of  all  living  beings. 
Closely  linked  on  the  one  hand  to  the  plant  kingdom  by  the  yeasts 
and  the  molds,  and  on  the  other  to  the  animal  kingdom  by  the  pro- 
tozoa, they  themselves  combine,  within  one  arid  the  same  division, 
attributes  so  widely  divergent  as  to  structure,  metabolism,  and  bio- 
logical activity  that  their  grouping  is  more  a  matter  of  working  con- 

29  Th.  Smith,  Jour,  of  Experimental  Med.,  No.  3,  1899. 
80  Certes,  Compt.  rend,  de  1'acad.  d.  sc.,  99,  Paris,  1884. 


40  BIOLOGY  AND  TECHNIQUE 

venience  than  of  actual  scientific  classification.  Thus,  for  instance, 
all  stages  of  metabolic  activity  fill  in  the  gap  between  the  synthetiz- 
ing  sulphur  and  nitrifying  bacteria  and  the  purely  katabolic  activ- 
ities of  some  of  the  aerobic  and  anaerobic  microorganisms  which 
cause  putrefaction.  Growth  takes  place  within  the  limits  of  a  wide 
temperature  range,  and  the  specific  modes  of  life  and  cultural  condi- 
tions are  subject  to  the  widest  variations,  from  those  of  an  indis- 
putably useful  saprophytism  to  those  of  the  most  exquisite  para- 
sitism. Although,  therefore,  strictly  speaking,  the  bacteria  can  be 
classified  as  a  whole  neither  in  the  animal  nor  in  the  vegetable 
realms,  being  nonchlorophyll-bearing,  they  are  for  convenience  clas- 
sified with  the  fungi  or  colorless  plants. 

The  relationship  of  the  bacteria  to  other  simple  plants  may  be 
graphically  represented  by  the  following  scheme: 

CRYPTOGAMIA 

THALLOPHYTA 

I 


ALGJE  LICHENS  FUNGI 

| 

SCHIZOMYCETES  BLASTOMYCETES  HYPHOMYCETES 

(Bacteria)  (Yeasts)  (Molds— Oidia) 

I 


Coccacea  Chlamydobacteria 

Bacteriaceae  (Higher  bacteria) 

Spirillaceae  Streptothrix 

Cladothrix 
Leptothrix 
Actinomyces 

The  special  classification  of  the  bacteria  has  offered  still  greater 
difficulties,  for  the  lower  we  proceed  in  the  phylo genetic  scale  of 
living  beings,  the  less  specialized  the  morphological  and  biological 
characteristics  of  any  group  become,  and  the  more  difficult  it  is  to 
establish  a  classification  which  can  in  any  way  be  regarded  as  final. 
It  is,  therefore,  quite  impossible  to  classify  the  bacterial  varieties  or 
species  on  any  basis  which  can  hope  to  satisfy  all  the  demands  of 
scientific  accuracy  and  it  is  necessary  to  resort  to  the  expedient  of 
utilizing  some  one  characteristic  which  remains  constant  for  the 
individual  genus  and  to  base  upon  this  an  attempt  at  grouping. 
When  bacteria  were  first  discovered,  and  for  many  years  following, 
numerous  observers  contended  that  the  form  of  the  microorganism 


RELATION  TO  ENVIRONMENT— CLASSIFICATION  41 

observed  was  not  a  constant  one  for  each  genus,  but  that  cocci  could 
be  converted  into  bacilli  or  spirilla  according  to  environmental  con- 
ditions. It  was  Cohn31  who,  in  1872,  first  recognized  the  constancy 
of  the  morphology  of  bacteria  and  established,  upon  morphological 
basis,  a  classification  which,  with  minor  changes,  has  been  retained 
until  the  present  day.  Such  classifications  can  not,  however,  be  re- 
garded as  anything  more  than  a  convenient  make-shift  pending  the 
day  when  the  finer  structure  and  true  biological  relations  of  the 
various  bacteria  shall  have  been  more  accurately  investigated.  The 
scheme  most  commonly  accepted  at  present  is  the  one  given  below, 
proposed  by  Migula : 32 

Bacteria  (Schizomycetes). — Fission  fungi  (chlorophyll  free),  cell  division  in 
one,  two,  or  three  directions  of  space.  Many  varieties  possess  power  of 
forming  endospores.  Whenever  motility  is  present,  it  is  due  to  flagella, 
or,  more  rarely,  to  undulating  membranes. 

FAMILY  I.   COCCACE^E. — Cells  in  free  state  spherical.    Division  in  one,  two, 

or  three  directions  of  space,  by  which  each  cell  divides  into  two,  four, 

or  eight  segments,  each  of  which  again  develops  into  a  sphere.     Endo- 

spore  formation  rare. 
Genus  I.   Streptococcus. — Cells  divide  in  one  direction  of  space  only,  for 

which  reason,  if  they  remain  connected  after  fission,  bead-like  chains 

may  be  formed.    No  organs  of  locomotion. 
Genus  II.   Micrococcus  (Staphylococcus). — Cells  divide  in  two  directions 

of  space,  whereby,  after  fission,  tetrad  and  grape-like  clusters  may  be 

formed.    No  organs  of  locomotion. 
Genus  III.   Sarcina. — Cells  divide  in  three  directions  of  space,  whereby, 

after  fission,  bale-like  packets  are  formed.    No  organs  of  .locomotion. 
Genus  IV.    Planococcus. — Cells  divide  in  two  directions  of  space,  as  in 

micrococcus,  but  possess  flagella. 
Genus  V.    Planosarcina. — Cells  divide  in  three  directions  o£  space  as  in 

sarcina,  but  possess  flagella. 

FAMILY  II.  BACTERIACE^:. — Cells  long  or  short,  cylindrical,  straight,  never 
spiral.  Division  in  one  direction  of  space  only,  after  preliminary 
elongation  of  the  rods. 

Genus  I.    Bacterium. — Cells  without  flagella,  often  with  endospores. 

Genus  II.  Bacillus. — Cells  with  peritrichal  flagella,  often  with  endo- 
spores. 

31  Cohn,  "Beitrage  zur  Biol.  d.  Pflanzen,"  Heft  1  u.  2,  1872. 
82 Migula,  " System  d.  Bakt.,"  Jena,  1897. 


42  BIOLOGY  AND  TECHNIQUE 

Genus  III.  Pseudomonas. — Cells  with  polar  flagella.  Endospores  occur 
in  a  few  species,  but  are  rare. 

FAMILY  III.   SPIRILLACE^B. — Cells  spirally  curved  or  representing  a  part  of 

a  spiral  curve.    Division  in  one  direction  of  space  only,  after  preceding 

elongation  of  cell. 

Genus  I.    Spirosoma. — Cells  without  organs  of  locomotion.     Rigid. 
Genus   II.    Microspira. — Cells  rigid,  with  one  or,  more  rarely,  two  or 

three  polar  undulated  flagella. 
Genus  III.    Spirillum. — Cells  rigid,  with  polar  tufts  of  five  to  twenty 

flagella  usually  curved  in  semicircular  or  flatly  undulating  curves. 
Genus  IV.    Spirochcete. — Cells  sinously  flexible.     Organs  of  locomotion 

unknown,  perhaps  a  marginal  undulating  membrane. 

FAMILY  IV.  CHLAMYDORBACTERIACE^E. — Forms  of  varying  stages  of  evolu- 
tion, all  possessing  a  rigid  sheath  (Hiille),  which  surrounds  the  cells. 
Cells  united  in  branched  or  unbranched  threads. 

Genus  I.  Streptothrix. — Cells  united  in  simple,  unbranched  threads. 
Division  in  one  direction  of  space  only.  Reproduction  by  non-motile 
conidia. 

Genus  II.  Cladothrix. — Cells  united  or  pseudodichotomously  branching 
threads.  Division  in  one  direction  of  space  only.  Vegetative  multipli- 
cation by  separation  of  entire  branches.  Reproduction  by  swarming 
forms  with  polar  flagella. 

Genus  III.  Crenothrix. — Cells  united  in  unbranched  threads,  at  first  with 
division  in  one  direction  of  space  only.  Later  the  cells  divide  in  all 
three  directions  of  space.  The  daughter  cells  become  rounded  and 
develop  into  reproductive  cells. 

Genus  IV.  Phragmidiothrix. — Cells  at  first  united  in  unbranched  threads, 
dividing  in  three  directions  of  space,  thus  forming  a  rope  of  cells. 
Later  some  of  the  cells  may  penetrate  through  sheath,  and  thus  give 
rise  to  branches. 

Genus  V.  Thiothrix. — Unbranched,  non-motile  threads,  inclosed  in  fine 
sheaths.  Division  of  cells  in  one  direction  only.  Cells  contain  sulphur 
granules. 

FAMILY  V.    BEGGIATOACE^E. — Cells  united  in  sheathless  threads.     Division 
in  one  direction  of  space  only.    Motility  by  undulating  membrane  as  in 
Oscillaria. 
Genus  Beggiatoa. — Cells  with  sulphur  granules. 

It  will  be  seen  in  reviewing  the  classification  just  given  that  the 
subdivisions  are  based  upon  questions  of  form,  motility,  and  situa- 
tion of  flagella.  While  these  characteristics,  so  far  as  we  know,  are 


RELATION   TO  ENVIRONMENT— CLASSIFICATION  43 

constant,  there  are,  nevertheless,  many  instances  in  which  types 
entirely  similar  in  these  respects  must  be  differentiated.  This  can  be 
done  only  by  careful  study  of  staining  reactions,  finer  structure, 
cultural  characteristics,  and  biological  activities. 

As  a  matter  of  fact,  while  the  botanical  classification  of  the  bac- 
teria offers  great  difficulties,  identification  is  not  so  complicated  a 
task  as  this  would  indicate.  Identification,  once  roughly  made  on  a 
morphological  basis,  is  further  carried  on  by  the  aid  of  cultural  char- 
acteristics, by  biochemical  reactions  and  by  pathogenic  properties. 
The  bacteria  occupy  so  important  a  place  in  agriculture,  in  medicine, 
and  in  hygiene,  that  it  rarely  becomes  necessary  for  a  worker  in  any 
particular  field  to  survey  the  entire  group.  The  habitat  of  a  large 
number  of  species  is  so  well  known  that  this  consideration  alone 
often  gives  a  clue  to  actual  identification. 

Bacterial  Mutation. — The  earlier  views  of  bacteriologists  con- 
cerning mutation  differed  greatly,  Naegeli  holding  that  extensive 
mutation  was  probably  the  rule;  Cohn,  on  the  other  hand,  holding 
strictly  to  the  constancy  of  form  and  species.  The  accumulated  ex- 
perience of  many  bacteriologists  during  the  years  since  then  seems 
to  point  almost  entirely  in  the  direction  indicated  by  Cohn,  and,  in 
fact,  most  of  our  methods  of  classification  are  based  upon  the  as- 
sumption of  such  constancy. 

Form  alone,  of  course,  cannot  be  relied  upon  for  classification 
among  organisms  so  simply  constructed  that  the  possibilities  of 
variation  in  form  are  very  limited.  In  classifying  bacteria,  there- 
fore, we  are  forced  to  take  cognizance  not  only  of  morphology,  but 
also  of  staining  characteristics,  behavior  on  differential  media,  fer- 
mentation reactions,  pathogenicity,  and,  as  a  final  appeal,  reactions 
with  specific  immune  sera.  The  last  especially,  as  utilized  in  agglu- 
tination and  complement-fixation,  seems  to  indicate  a  fundamental 
chemical  difference  in  the  constitution  of  bacteria  often  morpho- 
logically very  much  alike.  It  is  certainly  a  remarkable  fact  that 
organisms  such  as  those  belonging  to  the  colon-typhoid-dysentery 
group,  though  morphologically  not  differentiable,  may  still  retain 
differences  both  in  pathogenicity  and  in  fermentation  powers  after 
being  kept  for  ten  or  more  years  in  laboratory  media,  and  we  have 
had  the  same  experience  with  organisms  belonging  to  the  diphtheria 
group.  The  virulence  of  plague  and  anthrax  bacilli  may  be  retained 
for  years  in  storage,  and  such  evidence  shows  pretty  definitely  that 
fundamental  constant  differences  between  organisms  exist. 


44  BIOLOGY  AND  TECHNIQUE 

In  judging  of  mutation  we  must  differentiate  between  temporary 
changes  of  secondary  characteristics  which  revert  to  the  type  rapidly 
when  brought  back  to  the  normal  environment  and  those  which  con- 
stitute permanent  inherited  characteristics.  Of  recent  years  much 
work  has  been  done  on  this  question,  which  has  been  revived  very 
thoroughly  by  Eisenberg33  and  by  Vaughan.34  Systematic  cultiva- 
tion of  colon  and  typhoid  bacilli  in  the  hands  of  Twort,  Penfold  and 
others  seems  to  have  shown  that  agglutination  as  well  as  fermenta- 
tion characteristics  can  be  artificially  changed.  Furthermore,  color- 
producing  organisms  like  the  prodigiosus  can  be  artificially  changed 
to  colorless  strains,  and  it  is  well  known  that  certain  microorganisms 
rapidly  lose  their  virulence  when  cultivated,  and  that  the  virulence 
can  only  be  brought  back  by  passage  through  animals.  Rosenow  3; 
claims  recently  to  have  converted  hemolytic  streptococci  into  typical 
streptococcus  viridans,  pneumococcus  mucosus,  and  pneumococcus- 
like  organisms.  In  just  how  far  these  observations  will  be  shown 
to  represent  true  permanent  mutations  we  are  not  at  present  ready 
to  determine.  If  it  will  be  found  that  organisms  typically  repre- 
sentative of  a  well-known  species  can  be  changed  in  the  animal  body 
or  in  culture  into  forms  recognizedly  typical  of  another  species,  we 
will  have  to  revise  our  classifications,  and  we  can  look  upon  the 
classes  as  now  established  merely  as  convenient  methods  of  making 
discussion  possible,  but  not  as  representing  botanically  constant 
types. 

While  we  must  therefore  admit  that  a  considerable  degree  of 
mutation  is  possible,  we  do  not  ourselves  believe  that  the  evidence 
is  sufficiently  strong  to  undermine  the  prevailing  ideas  as  to  the 
constancy  of  species.  Most  mutations  so  far  produced  have  readily 
reverted  to  type  when  subjected  to  proper  conditions. 


™  Eisenberg,  Weichhardt's  Ergebnesse,  1914. 

M  Vaughan,  Jour,  of  Lab.  &  Clin.  Met!.,  1915,  vol.  1,  145. 

KKosenow,  Jour.  Infect.  Dis.,  xiv,  1914,  1. 


CHAPTER  IV 

THE   BIOLOGICAL  ACTIVITIES   OF  BACTERIA 

WHILE  the  bacteria  pathogenic  to  man  and  animals  largely  usurp 
the  attention  of  those  interested  in  disease  processes,  this  group  of 
microorganisms  is  after  all  but  a  small  specialized  off-shoot  of  the 
realm  of  bacteria,  and,  broadly  speaking,  actually  of  minor  impor- 
tance. Surveying  the  existing  scheme  of  nature,  as  a  whole,  it  is  not 
an  extravagant  statement  to  say  that  without  the  bacterial  processes 
which  are  constantly  active  in  the  reduction  of  complex  organic  sub- 
stances to  their  simple  compounds,  the  chemical  interchange  between 
the  animal  and  vegetable  kingdoms  would  fail,  and  all  life  on  earth 
would  of  necessity  cease.  To  understand  the  full  significance  of 
this,  it  is  necessary  to  consider  for  a  moment  the  method  of  the 
interchange  of  matter  between  the  animal  and  vegetable  kingdoms. 

All  animals  require  for  their  sustenance  organic  compounds. 
They  are  unable  to  build  up  the  complex  protoplasmic  substances 
which  form  their  body  cells  from  chemical  elements  or  from  the 
simple  inorganic  salts.  They  are  dependent  for  the  manufacture  of 
their  food-stuffs,  therefore,  directly  or  indirectly,  upon  the  synthetic 
or  anabolic  activities  of  the  green  plants. 

These  plants,  by  virtue  of  the  chlorophyll  contained  within  the 
cells  of  their  leaves  and  stems,  and  under  the  influence  of  sunlight, 
possess  the  power  of  utilizing  the  carbon  of  the  carbonic  acid  gas 
of  the  atmosphere,  and  of  combining  it  with  water  and  the  nitro- 
genous salts  absorbed  by  their  roots,  building  up  from  these  simple 
radicles  the  highly  complex  substances  required  for  animal  susten- 
ance. 

These  products  of  the  synthetic  activity  of  the  green  plants,  then, 
are  ingested  by  members  of  the  animal  kingdom,  either  directly,  in 
the  form  of  vegetable  food,  or  indirectly,  as  animal  matter.  They 
are  utilized  in  the  complex  laboratory  of  the  animal  body  and  are 
again  broken  down  into  simpler  compounds,  which  leave  the  body 
as  excreta  and  secreta. 

The  excreta  and  secreta  of  animals,  however,  are,  in  a  small  part 

45 


46  BIOLOGY  AND  TECHNIQUE 

only,  made  up  of  substances  simple  enough  to  be  directly  utilized 
by  plants.  The  dead  bodies,  moreover,  of  both  animals  and  plants 
would  be  of  little" further  value  as  stores  of  matter  unless  new  factors 
intervened  to  reduce  them  to  that  simple  form  in  which  they  may 
again  enter  into  the  synthetic  laboratory  of  the  green  plant.  Agents 
for  further  cleavage  of  these  compounds  are  required,  and  these  are 
supplied  by  the  varied  activities  of  the  bacteria. 

On  the  other  hand,  bacteria  are  also  important  in  the  process  of 
synthesis.  The  main  supply  of  nitrogen  available  for  plant  life  is 
found  in  the  elementary  state  in  the  atmosphere — a  condition  in 
which  it  can  not  be  utilized  as  a  raw  product  by  the  plant.  This  gap 
again  is  bridged  by  the  bacteria  found  in  the  root  bulbs  of  the  legu- 
minous plants — bacteria  which  possess  the  power  of  assimilating  or 
aiding  in  the  assimilation  of  atmospheric  nitrogen  and  its  prepara- 
tion for  further  use  by  the  plant  itself.  Another  bacterial  activity 
which  may  be  classified  as  an  anabolic  process  is  the  oxidation  of 
the  ammonia,  released  by  decomposition,  into  nitrites  and  nitrates. 
This  is  carried  on  by  certain  bacteria  of  the  soil.  These  are  to  be 
treated  of  in  greater  detail  in  another  section. 

There  is  a  constant  circulation,  therefore,  of  nitrogen  and  carbon 
compounds,  between  the  plant  and  the  animal  kingdoms,  by  virtue 
of  an  anabolic  or  constructive  process  in  the  one,  and  a  katabolic  or 
destructive  process  in  the  other,  rendering  them  mutually  interde- 
pendent and  indispensable.  The  circuit,  however,  is  not  by  any 
means  a  closed  one ;  there  are  important  gaps,  both  in  the  process  of 
cleavage  and  in  that  of  synthesis,  which,  if  left  unbridged  by  the 
bacteria,  would  effectually  arrest  all  life-activity  of  plants  and 
eventually  of  animals. 

Far  from  being  scourges,  therefore,  these  minute  microorganisms 
are  paramount  factors  in  the  great  cycle  of  living  matter,  supplying 
necessary  links  in  the  circulation  of  both  nitrogenous  and  carbon 
compounds. 

KATABOLIC  ACTIVITIES  OF  BACTERIA 

The  katabolic  activities  of  bacteria,  then,  consist  in  the  fermenta- 
tion of  carbohydrates  and  in  the  cleavage  of  proteins  and  fats. 

Fermentation  is  carried  out  to  a  large  extent  by  the  yeasts,  but 
also  to  no  inconsiderable  degree  by  bacteria.  Protein  decomposition 
and  the  cleavage  of  fats  are  carried  out  almost  exclusively  by 
bacteria. 


THE   BIOLOGICAL  ACTIVITIES  OF   BACTERIA  47 

For  our  knowledge  of  the  fundamental  laws  underlying  these 
phenomena  of  fermentation  and  protein  decomposition,  we  are  in- 
debted to  the  genius  of  Pasteur,1  who  was  the  first  to  prove  experi- 
mentally the  exclusive  and  specific  parts  played  by  various  microor- 
ganisms in  these  processes.  While  the  observations  and  deductions 
made  by  Pasteur  have  not  been  greatly  modified,  a  large  store  of  in- 
formation has  been  gained  since  his  time,  which  has  thrown  addi- 
tional light  upon  the  chemical  details  and  the  more  exact  manner  of 
action  of  the  factors  involved. 

The  actual  work  of  cleavage  in  both  fermentation  and  protein 
cleavage  is  carried  out  by  substances  known  as  enzymes  or  ferments, 
the  nature  of  which  we  must  further  discuss  before  their  manner  of 
action  can  be  fully  comprehended. 

Bacterial  Enzymes  or  Ferments. — A  ferment  or  enzyme  is  a  sub- 
stance produced  by  a  living  cell,  which  brings  about  a  chemical  re- 
action without  entering  into  the  reaction  itself.  The  enzyme  itself  is 
not  bound  to  any  of  the  end  products  and  is  not  appreciably  dimin- 
ished in  quantity  after  the  reaction  is  over,  although  its  activity  may 
be  finally  inhibited  by  one  or  another  of  the  new  products.  The  action 
of  bacterial  enzymes  is  thus  seen  to  be  closely  similar  to  that  of  the 
chemical  agents  technically  spoken  of  as  ' Catalyzers,"  represented 
chiefly  by  dilute  acids.  Thus,  if  an  aqueous  solution  of  saccharose  is 
brought  into  contact  with  a  dilute  solution  of  sulphuric  acid,  the  di- 
saccharid  is  hydrolyzed  and  is  decomposed  into  levulose  and  dextrose. 

Thus : 

C^H^O,,    +    H20          C6H1206    +  C6H120G 
In  contact  with  Dextrose         Levulose 

dilute  H2S04 

During  this  process,  which  is  known  as  ''inversion,"  the  concentra- 
tion of  the  sulphuric  acid  remains  entirely  unchanged.  While  theo- 
retically the  changes  brought  about  by  enzymes  and  katalyzers  are 
usually  such  as  would  occur  spontaneously,  the  time  for  the  sponta- 
neous occurrence  would  be,  at  ordinary  temperatures,  infinitely  long. 
The  definition  for  enzymes  and  katalyzers  is  given  by  Ostwald,  there- 
fore, as  "substances  which  hasten  a  chemical  reaction  without  them- 
selves taking  part  in  it. ' '  Exactly  the  same  result  which  is  obtained 
by  the  use  of  dilute  sulphuric  acid  is  caused  by  the  ferment  "inver- 
tasc"  produced,  for  instance,  by  B.  megatherium.  Were  a  solution 

1  Pasteur,  ''Etude  sur  la  biere,"  Paris,  1876. 


48  BIOLOGY  AND  TECHNIQUE 

of  saccharose  subjected  to  heat,  without  kacalyzer  or  ferment,  a 
similar  change  would  occur,  but  by  the  mediation  of  these  substances 
the  inversion  is  produced  without  other  chemical  or  physical  rein- 
forcement. 

This  analogy  between  enzymes  and  katalyzing  agents  is  very 
striking.  Thus,  as  stated,  both  katalyzers  and  enzymes  bring  about 
changes  without  themselves  being  used  up -in  the  process,  both  act 
without  the  aid  of  heat,  and  the  reactions  brought  about  by  both 
have  occasionally  been  shown  to  be  reversible.  While  this  last  phe- 
nomenon has  been  variously  shown  for  katalyzers,  the  process  of 
reversibility  has  been  demonstrated  for  bacterial  enzyme  action  only 
in  isolated  cases.  Thus,  it  has  been  found  that  by  the  action  of  the 
yeast  enzyme  maltase  upon  concentrated  dextrose  solutions,  a  re- 
formation of  maltose  may  occur.  In  both  cases,  moreover,  the  quan- 
tity of  enzyme  or  katalyzer  is  infinitely  small  in  proportion  to  the 
amount  of  material  converted  by  their  action. 

There  is  a  close  similarity,  furthermore,  between  the  bacterial  en- 
zymes and  the  ferments  produced  by  specialized  cells  of  the  higher 
animals  and  plants.  For  instance,  the  action  of  the  ptyalin  of  the 
saliva  or  of  the  diastase  obtained  from  plants  is  entirely  analogous  to 
the  starch-splitting  action  of  the  amylase  produced  by  many  bacteria. 

The  action  of  all  enzymes  depends  most  intimately  upon  environ- 
mental conditions.  For  all  of  them  the  presence  of  moisture  is 
essential.  All  of  them  depend  for  the  development  of  their  activity 
upon  the  existence  of  a  specifically  suitable  reaction.  Strong  acids 
or  alkalies  always  inhibit,  often  destroy  them.  Temperatures  of  over 
70°  C.  permanently  destroy  most  enzymes,  whereas  freezing,  while 
temporarily  inhibiting  their  action,  causes  no  permanent  injury,  so 
that  upon  thawing,  their  activity  may  be  found  almost  unimpaired. 
Direct  sunlight  may  injure,  but  rarely  destroys,  ferments.  Against 
the  weaker  disinfectants  in  common  use,  enzymes  often  show  a  higher 
resistance  than  do  the  bacteria  which  give  rise  to  them. 

The  optimum  conditions  for  enzyme  action,  then,  consist  in  the 
presence  of  moisture,  the  existence  of  a  favorable  reaction,  weakly 
acid  or  alkaline,  as  the  case  may  be,  and  a  temperature  ranging  from 
35°_45°  c.2 

Proteolytic  Enzymes. — In  nature,  the  decomposition  of  dead  ani- 
mal and  vegetable  matter  occurs  only  when  the  conditions  are  favor- 


-OppenJieimer,  "Die  Fermente,"  etc.    Leipzig,  1900. 


THE   BIOLOGICAL  ACTIVITIES  OF  BACTERIA  49 

able  for  bacterial  development.  Thus,  as  is  well  known,  freezing, 
sterilizing  by  heat,  or  the  addition  of  disinfectants  will  prevent  the 
rotting  of  organic  material. 

In  the  laboratory,  the  presence  of  proteolytic  enzymes  is  deter- 
mined chiefly  by  the  power  of  bacteria  to  liquefy  gelatin,  fibrin,  or 
coagulated  blood  serum.  These  ferments  are  not  always  secretions 
from  the  bacterial  cell,  but  in  some  cases  may  be  closely  bound  to 
the  cell-body  and  separable  only  by  extraction  after  death.  In  such 
cases  they  are  spoken  of  as  endoenzymes.  Whenever  they  are  true 
secretory  products,  however,  they  can  be  obtained  separate  from 
the  microorganisms  which  form  them  by  filtration  through  a  Berke- 
feld  candle.  From  such  filtrates  they  may,  in  some  cases,  be  obtained 
in  the  dry  state  by  precipitation  with  alcohol.  When  obtained  in 
this  way  the  precipitated  enzyme  is  usually  much  more  thermostable 
than  when  in  solution,  for  while  soluble  enzymes  in  filtrates  are 
usually  destroyed  by  70°  C.,  and  even  less,  the  dried  powder  may 
occasionally  withstand  140°  C.  for  as  long  as  ten  minutes.3 

Apart  from  the  general  conditions  of  temperature  and  moisture, 
the  development  of  these  enzymes  seems  to  depend  directly  upon  the 
presence  of  proteins  in  the  culture  media.  The  number  of  bacterial 
species  which  produce  proteolytic  enzymes  is  legion.  Among  those 
more  commonly  met  with  are  staphylococci,  B.  subtilis,  B.  proteus,  B. 
faecalis  liquefaciens,  Spirillum  choleras  asiaticae,  B.  anthracis,  B. 
tetani,  B.  pyocyaneus,  and  a  large  number  of  others.  The  inability 
of  any  given  microorganism  to  liquefy  gelatin  or  fibrin  by  no  means 
entirely  excludes  the  formation  by  it  of  proteolytic  enzymes,  since 
these  ferments  may  often  be  active  for  one  particular  class  of  protein 
only. 

In  order  to  study  the  qualitative  and  quantitative  powers  of  any 
given  bacterial  proteolyzing  enzyme  or  protease,  it  is,  of  course, 
necessary  to  study  these  processes  in  pure  culture  in  the  test  tube 
with  media  of  known  composition.  In  the  refuse  heap,  in  sewage,  or 
in  rotting  excreta,  the  process  is  an  extremely  complicated  one,  for 
besides  the  bacteria  which  attack  the  protein  molecule  itself,  there 
are  many  other  species  supplementing  these  and  each  other,  one 
species  attacking  the  more  or  less  complex  end-products  left  by  the 
action  of  the  others. 

Exactly  what  the  chemical  reactions  are  which  take  place  in  these 


*  Fuhrmann,  "Die  Bakterienzyme, ' ;  p.  45. 


50  BIOLOGY  AND  TECHNIQUE 

cleavages  is  not  entirely  clear.  It  is  believed,  however,  that  most  of 
the  cleavages  are  of  an  hydrolytic  nature. 

In  general,  the  action  of  the  protein-splitting  ferments  is  com- 
parable to  that  of  the  pancreatic  ferment  trypsin,  and  they  arc  most 
often  active  in  an  alkaline  environment.  They  differ,  among  them- 
selves, in  the  extent  to  which  they  are  able  to  reduce  the  protein 
molecule  to  its  simple  radicles,  some  types  leading  to  a  relatively 
mild  cleavage,  while  others,  of  the  proteus  type,  yield  ammo  acids. 
Many  species  of  bacteria  which  are  unable  to  secrete  a  proteolytic 
enzyme,  nevertheless  produce  erepsin-like  ferments  which  readily 
attack  peptones  or  polypeptides,  with  the  formation  of  amino  acids. 

A  distinction  is  occasionally  made  between  the  terms  putrefaction 
and  decay,  the  former  being  used  to  refer  to  the  decomposition 
taking  place  under  anaerobic  conditions,  that  is,  in  the  absence  of 
oxygen,  a  process  resulting  in  the  production  of  amino  acids,  H2S, 
indole,  skatole  and  particularly  the  mercaptans,  which  cause  the 
highly  offensive  odor  characteristic  of  this  type  of  decomposition. 
The  gases  generated  in  such  decomposition  are  largely  made  up  of 
C02  and  hydrogen.  The  coincident  presence,  furthermore,  of  the 
carbohydrate-splitting  bacteria  and  of  denitrifying  microorganisms 
renders  the  actual  process  of  putrefaction  a  chaos  of  many  activities 
in  which  the  end-products  and  by-products  are  qualitatively 
determinable  only  with  little  precision,  and  which  completely 
defies  any  attempt  at  quantitative  analysis.  Decay  is  the  term  used 
to  signify  decomposition  under  aerobic  conditions,  leading  primarily 
to  the  formation  of  amino  acids,  which  are  as  a  rule,  changed  further 
to  carbon  dioxide,  water  and  ammonia,  often  together  with  H2S  and 
less  completely  decomposed  substances,  such  as  indole  and  various 
amines.  Mercaptans  are  never  formed  and  the  foul  odor  of  putre- 
faction is  not  present  in  this  aerobic  proteolysis.4 

Ptomaines. — Very  early  in  the  study  of  the  products  of  bacterial 
growth,  a  number  of  well  defined  crystalline  basic  substances  were 
isolated  from  protein  material  which  had  undergone  bacterial  putre- 
faction. These  received,  as  a  class,  the  name  of  ptomaines  (from 
TTTto/Aa,  a  dead  body),  and  were  shown  to  be  toxic  when  fed  to 
or  injected  into  animals.  It  was  attempted,  at  the  time  of  their 
discovery,  to  explain  the  action  of  pathogenic  bacteria  on  the  basis 
of  a  production  of  ptomaines  or  related  substances.  This  soon 


4  Eettger  and  Newell,  Jour,  of  Biol.  Chem.,  xii,  1912,  341. 


THE  BIOLOGICAL  ACTIVITIES   OF  BACTERIA  51 

proved  not  to  be  the  case,  for  not  only  were  these  bases  never  toxic 
in  the  minute  dose  sufficient  for  the  true  toxins  of  diphtheria  and 
tetanus,  but  the  anatomical  lesions  produced  were  different.  More- 
over, true  bacterial  toxins  seemed  to  be  formed  to  a  great  extent 
independent  of  the  composition  of  the  media  upon  which  growth 
was  obtained,  while,  as  will  be  seen,  ptomaine  production  depends 
directly  upon  the  constitution  of  the  substrate.  Finally,  nothing  in 
the  nature  of  antitoxin  production  could  be  shown,  following  suble- 
thal  injections  of  the  ptomaines.  While  it  is,  therefore,  not  possible 
to  account  for  the  symptoms  of  bacterial  action  in  the  body  on  the 
basis  of  ptomaines,  it  is  not  impossible  that  the  ptomaines  them- 
selves, if  ingested  in  food  which  has  undergone  some  putrefactive 
changes,  may  occasionally  cause  sickness.  Food  poisoning  of  this 
variety  was  formerly  believed  to  be  very  common,  and  was  described 
under  various  names,  as  kreatoxismus  (meat  poisoning) ,  tyrotoxismus 
(cheese  poisoning)  and  sitotoxismus  (vegetable  poisoning).  At 
present  there  is  much  question  as  to  whether  food  poisoning, 
wherever  met  with  (excepting,  of  course,  botulism,  which  is  caused 
by  the  ingestion  of  a  true  bacterial  toxin),  is  not  caused  by  the 
presence  of  living  bacteria  of  the  Gartner  type  in  the  food,  and  not 
to  ptomaines.5  It  is  quite  possible,  on  the  other  hand,  that  bacteria 
present  in  the  intestine  may,  under  certain  conditions,  set  up  putre- 
factive changes  in  the  large  bowel,  leading  to  the  formation  of 
.  ptomaines  or  closely  allied  substances  which  can  be  absorbed  directly 
and  cause  illness. 

Chemically,  the  ptomaines  are  bases  produced  by  the  splitting 
out  of  C02  from  the  acid  group  of  the  amino  acids.  Amines  have 
been  isolated  resulting  from  this  form  of  decomposition  of  practically 
all  the  known  amino  acids.  Of  historic  interest  are  putrescine  and 
cadaverine.  The  former  is  produced  by  the  decarboxylation  of 
ornithine,  thus: 

CH2  (NH2)  CH2  •  CH2  -  CH  (NH2)  COOH  = 

CH2(NH2)  •  CH2  •  CH2  •  CH2(NH2) +CO2 

Cadaverine  is  similarly  formed  from  lysine.  Both  these  bases 
possess  relatively  slight  toxicity.  Much  more  highly  toxic  is  the 
base  histamine,  obtained  in  an  entirely  analogous  manner  from  his- 


5  Jordan,  "Food  Poisoning/'  University  of  Chicago  Press,  1917. 


52  BIOLOGY  AND  TECHNIQUE 

tidine.  Some  light  seems  to  have  been  shed  on  the  mechanism  of 
this  process  of  decarboxylizatioii  by  Koessler  and  Hanke,*5  who 
found,  while  investigating  the  production  of  histamine  by  B.  coli, 
that  it  was  formed  only  in  cultures  in  which  considerable  quantities 
of  acid  were  being  produced  at  the  same  time,  and  they  believe 
that  the  production  of  this  strongly  basic  substance  offsets  to  a 
degree  the  acid  production  and  enables  the  organisms  to  develop 
further  before  the  H-ion  concentration  reaches  the  point  of  inhibi- 
tion. Sepsine,  another  base  of  unknown  composition,  but  probably 
related  in  structure  to  the  other  ptomaines,  has  been  isolated  by 
Faust  from  putrid  yeast,  and  has  been  shown  to  be  highly  toxic. 

In  breaking  down  animal  excreta,  the  task  of  the  bacteria  is 
rather  a  simpler  one  than  when  dealing  with  the  cadavers  them- 
selves, for  here  a  part  of  the  cleavage  has  already  been  carried  out 
either  by  the  destructive  processes  accompanying  metabolism,  or  by 
partial  decomposition  by  bacteria  begun  within  the  digestive  tract. 
This  material  outside  of  the  body  is  further  reduced  by  bacterial 
enzymes  into  still  simpler  substances,  the  nitrogen  usually  being 
liberated  in  the  form  of  ammonia.  One  example  of  such  an  am- 
moniacal  fermentation  may  be  found  in  the  case  of  the  urea  fermen- 
tation by  Micrococcus  urea?,  in  which  the  cleavage  of  the  urea  takes 
place  by  hydrolysis  according  to  the  following  formula: 

(NH,)2  CO  +  2H,  0  =  C02  +  2NH3  +  H2  0 

Similar  ammoniacal  fermentations  are  carried  out,  though  perhaps 
according  to  less  simple  formulae,  by  a  large  number  of  microor- 
ganisms. Perhaps  the  most  common  species  which  possesses  the 
power  is  the  group  represented  by  B.  proteus  vulgaris  (Hauser). 

From  what  has  been  said  it  follows  naturally  that,  so  far,  the 
decomposition  of  the  protein  molecule  from  its  complex  structure 
to  ammonia  or  simple  ammonia  compounds  is  an  indispensably  im- 
portant function,  not  only  for  agriculture,  but  for  the  maintenance 
of  all  life  processes.  It  is  clear,  on  the  other  hand,  that  a  further 
decomposition  of  ammonia  compounds  into  forms  too  simple  to  be 
utilized  by  the  green  plants  would  be  a  decidedly  harmful  activity. 
And  yet  this  is  brought  about  by  the  so-called  denitrifying  bacteria 
which  will  be  considered  in  a  subsequent  section. 

8  Koessler  and  HanTce,  J.  Biol.  Chem.,  1919,  39,  539. 


THE  BIOLOGICAL  ACTIVITIES  OF  BACTERIA  53 

Lab  Enzymes. — There  are  a  number  of  ferments  produced  by 
bacteria  which,  although  affecting  proteins,  can  not  properly  be 
classified  with  the  proteolytic  enzymes.  These  are  the  so-called 
coagulases  or  lab  enzymes,  which  have  the  power  of  producing 
coagulation  in  liquid  proteins.  Just  what  chemical  process 
underlies  this  coagulation  is  not  known.  If  Hammarsten 's7 
conclusions  as  to  the  hydrolytic  nature  of  the  changes  produced 
by  them  are  true,  these  enzymes  are  brought  into  close  relationship 
to  the  proteolyzers,  although  a  coagulation  can  hardly  be  regarded 
as  a  true  katabolic  process.  In  milk  where  the  lab-action  becomes 
evident  by  precipitation  of  casein,  a  strict  differentiation  must  be 
made  between  this  coagulation  and  that  brought  about  by  acids  or 
alkalies.  In  the  former  case,  casein  is  not  only  precipitated  and 
converted  into  paracasein,  but  is  actually  changed  so  that  when 
redissolved  it  is  no  longer  precipitated  by  lab.8 

Coagulating  enzymes  for  milk  proteins,  blood,  and  other  protein 
solutions  are  produced  by  a  large  variety  of  bacteria.  They  have 
been  observed  in  cultures  by  the  cholera  vibrio,  B.  prodigosus,  B. 
pyocyaneus,  and  several  others.9 

The  lab  enzymes  are  easily  destroyed  by  temperatures  of  70°  C. 
and  over,  and  are  very  susceptible  to  excessive  acidity  or  alkalinity. 

Fat-Splitting  Enzymes  (Lipase). — The  fat-splitting  powers  of 
bacteria  have  been  less  studied  than  some  of  the  other  bacterial 
functions  and  are  correspondingly  more  obscure.  It  is  known,  never- 
theless, that  the  process  is  due  to  an  enzyme  and  that  it  is  probably 
hydrolytic  in  nature.  The  following  formula  represents  the  simplest 
method  in  which  some  of  the  molds  and  bacteria  produce  cleavage 
of  fats  into  glycerin  and  fatty  acid: 

C3  H5  (CnH2n-i  02)3  +  3H2  O  =  C3  H5  (OH3)  +  3Cn  H2n  O2 

Glycerin          Fatty  acid 

Some  of  the  bacteria  endowed  with  the  power  of  producing 
lipase  are  the  cholera  spirillum,  B.  fluorescens  liquefaciens,  B. 
prodigiosus,  B.  pyocyaneus,  Staphylococcus  pyogenes  aureus,  and 
some  members  of  the  streptothrix  family.  The  methods  of  in- 


7  Hamm  first  ctt,  ' '  Textbook  of  Physiol.  Chemistry,"  Translation  by  Mamlel. 
s  Oppenheimcr,  "Dip  I'Yrmoiilo  u.  ihre  Wirknng,"  Leipzig,  1903. 
9  Torini,  Atti  dei  laborat.  d.  sanita,  Rome,  1890. 


54  BIOLOGY  AND  TECHNIQUE 

vestigating  this  function  of  bacteria,  originated  by  Ejkniami,10  con- 
sists in  covering  the  bottom  of  a  Petri  dish  with  tallow  and  pouring 
over  this  a  thin  layer  of  agar.  Upon  this,  the  bacteria  are  planted. 
Any  diffusion  of  lipase  from  the  bacterial  colonies  becomes  evident 
by  a  formation  of  white,  opaque  spots  in  the  tallow.  Carriere11  was 
able  to  demonstrate  a  fat-splitting  ferment  for  the  tubercle  bacillus. 
Apart  from  the  importance  of  these  enzymes  in  nature  for  the 
destruction  of  fats,  they  are  industrially  important  because  of  their 
action  in  rendering  butter,  milk,  tallow,  and  allied  products  rancid, 
and  are  medically  of  interest  for  their  action  upon  fats  in  the  intes- 
tinal canal. 

Enzymes  of  Fermentation  (The  Cleavage  of  Carbohydrates  by 
Bacteria). — The  power  to  assimilate  carbon  dioxide  from  the 
atmosphere  is  possessed  only  by  the  green  plants  and  some  of  the 
colored  alga?,  and  the  sulphur  or  thiobacteria.  All  other  living 
beings  are  thus  dependent  for  their  supply  of  carbon  upon  the 
synthetic  activities  carried  on  by  these  plants  to  the  same  degree 
in  which  they  are  dependent  upon  similar  processes  for  their  nitrogen 
supply.  The  return  of  this  carbon  to  the  atmosphere  is,  of  course, 
brought  about  to  a  large  extent  by  the  respiratory  processes  of  the 
higher  animals.  The  carbon,  which,  together  with  nitrogen,  forms 
a  part  of  protein  combinations,  is  freed,  as  we  have  seen  in  a  previous 
section,  by  the  processes  of  protein  cleavage.  That,  however,  which 
is  inclosed  in  the  carbohydrate  molecule,  is  set  free  by  the  action 
of  yeasts,  molds,  or  bacteria,  by  an  enzymatic  process  similar  in 
every  respect  to  that  described  above  for  the  process  of  protein 
cleavage. 

FERMENTATION. — The  power  of  carbohydrate  cleavage  is  possessed 
by  a  large  number  of  the  yeasts  and  bacteria.  The  process,  as  has 
been  indicated,  is  of  great  importance  in  the  cycle  of  carbon  com- 
pounds for  the  return  of  carbon  to  its  simplest  forms,  and  is,  further- 
more, as  will  be  seen  in  a  later  section,  of  great  utility  in  the  indus- 
tries. In  each  case  the  power  to  split  a  particular  carbohydrate 
is  a  more  or  less  specific  characteristic  of  a  given  species  of  micro- 
organism, and  for  this  reason  has  been  extensively  used  as  a  method 
for  the  biological  differentiation  of  bacteria.  In  the  course  of  much 
careful  work  upon  this  question  it  has  been  ascertained  that  the 


™Ejkmann,  Cent.  f.  Bakt.,  I,  xxix,  1901. 

11  Carriere,  Comptes  rend,  de  la  soc.  de  biol.,  53,  1901. 


THE   BIOLOGICAL  ACTIVITIES  OF  BACTERIA  55 

specific  carbohydrate-splitting*  powers  of  any  given  species  are  con- 
stant and  unchanged  through  many  generations  of  artificial  cultiva- 
tion. Thus,  differentiation  of  the  Gram-negative  bacteria,  the 
diphtheria  group,  and  to  some  extent  of  the  members  of  the  pneu- 
mococcus-streptococcus  group,  can  now  largely  be  made  by  a  study 
of  their  sugar'  fermentations. 

In  most  of  these  cases,  as  far  as  we  know,  the  cleavage  is  produced 
by  a  process  of  hydrolysis.  A  convenient  nomenclature  which  has 
been  adapted  for  the  designation  of  these  ferments  is  that  which 
employs  the  name  of  the  converted  carbohydrate  adding  the  suffix 
"asc"  to  indicate  the  enzyme.  There  are  thus  ferments  known  as 
amylase,  cellulase,  lactase,  etc. 

Amylase  (Diastase  or  Amylolytic  Ferment). — Amylases  or  starch- 
splitting  enzymes  are  formed  by  many  plants  (malt)  and  by  animal 
organs  (pancreas,  saliva,  liver).  Among  microorganisms  amylase 
is  produced  by  many  of  the  streptothrix  group,  by  the  spirilla  of 
Asiatic  cholera  and  of  Finkler-Prior,  by  B.  anthracis,  and  many 
other  bacteria.  A  large  number  of  the  bacteria  found  in  the  soil, 
furthermore,  have  been  shown  to  produce  amylases.  By  cultivating 
bacteria  upon  starch-agar  plates,  amylase  can  be  readily  demon- 
strated by  a  clearing  of  the  medium  immediately  surrounding  the 
colonies.12 

Since,  of  course,  there  are  several  varieties  of  starches,  it  follows 
that  the  exact  chemical  action  of  amylase  differs  in  individual  cases. 
The  determination  of  the  structural  disintegration  of  starch  by  these 
ferments  is  fraught  with  much  difficulty,  owing  to  the  polymeric 
constitution  of  the  starches.  Primarily,  however,  a  cleavage  takes 
place  into  a  disaccharid  such  as  maltose  (hexobiose),  and  the  non- 
reducing  sugars  and  dextrin.  Beyond  this  point,  however,  the 
further  cleavages  are  subject  to  much  variation  and  are  not  entirely 
clear.  The  dextrins  upon  further  reduction  yield  eventually  maltose, 
and  this  in  turn,  dextrose. 

Another  most  interesting  example  of  amylolytic  activity  is  the 
fermentation  of  starch  by  such  organisms  as  the  B.  Granulobacter 
Pectinovorum  with  the  production  of  acetone  and  butyl  alcohol. 
This  reaction  is  now  used  industrially  for  the  preparation  of  acetone, 
using  a  corn  meal  mash  as  the  substrate.  Dextrose  is  first  formed, 
and  from  this,  acetone,  butyl  alcohol,  acetic  and  butyric  acid,  to- 


12  Ejkmann,  Cent.  f.  Bakt.,  xxix,  1901,  and  xxxv,  1904. 


56  BIOLOGY  AND  TECHNIQUE 

gether  with  hydrogen  and  C02  are  produced.  The  chemical 
mechanism  of  the  fermentation  is  thus  seen  to  be  highly  complex.13 
Cellulose. — Cellulose  is  fermented  by  a  limited  number  of  bacteria, 
most  of  them  anaerobes.  The  chemical  process  by  which  this  takes 
place  is  but  poorly  understood.14 

Gelase. — An  agar-splitting  ferment  has  been  found  by  Gran.15 
Invertase. — The  enzymes  which  hydrolytically  cause  cleavage  of 
saccharose  into  dextrose  and  levulose  are  numerous.     The  chemical 
process  takes  place  according  to  the  following  formula: 

C12  H22  On  +  H2  0  =  C6  H12  06  -f  C6  H12  06 
Saccharose  Dextrose       Levulose 

Invertase  is  produced  by  many  of  the  yeasts.  It  is  one  of  the  most 
common  of  the  enzymes  produced  by  bacteria,  and  has  been  found  in 
cultures  of  B.  megatherium,  B.  subtilis,  pneumococcus,  some  strepto- 
cocci, B.  coli,  and  many  others.  Invertase  is  usually  very  susceptible 
to  heat,  being  destroyed  by  temperatures  of  70°  C.  and  over.  A 
slightly  acid  reaction  of  media  abets  the  inverting  action  of  these 
enzymes.  Strong  acids  and  alkalies  inhibit  them.  Inverting  enzymes 
may  be  precipitated  out  of  solution  by  alcohol.  Antiseptics  even 
in  weak  concentrations  will  inhibit  their  action. 

Lactase. — Lactose-splitting  ferments  are  extremely  common  both 
among  bacteria  and  among  the  yeasts.  The  process  is  here  again  a 
hydrolytic  cleavage  resulting  in  the  formation  of  the  monosaccharids, 
dextrose  and  galactose. 

Maltase. — A  maltose-splitting  ferment  has  also  been  found  in  the 
cultures  of  many  bacteria,  leading  to  the  formation  of  dextrose. 

Lactic  Acid  Fermentation. — Lactic  acid  (oxyproprionic  acid, 
C3  HG  08)  is  one  of  the  -most  common  substances  to  appear  among 
the  products  of  bacterial  activity,  both  in  media  containing  carbohy- 
drates and  in  those  consisting  entirely  of  albuminous  substances. 
In  most  of  these  cases,  the  lactic  acid  is  formed  merely  as  a  by- 
product accompanying  many  other  more  complicated  chemical 
cleavages.  In  some  instances,  however,  lactic  acid  is  produced  from 
carbohydrates,  both,  disaccharids  and  monosaccharids,  as  an  almost 


"SpeaJcma-n,  Jour,  of  Biol.  Chem.,  xli,  1920,  319;  xliii,  1920,  401. 
^OmeliansM,  Lafar's  "Handb.  d.  techn.  Mykologio, "  Bd.  iii,  Chap.  9. 
15  Gran,  Bergens  Museum  Aarbog,  1902,  Hft.  I. 


THE  BIOLOGICAL  ACTIVITIES  OF  BACTERIA  57 

pure  product  due  to  a  specific  bio-chemical  process.  The  reactions 
taking  place  in  this  phenomenon  may  be  briefly  expressed  according 
to  the  following  formulae  : 


Lactose  Lactic  acid 

or 

CG  H12  0,  =  2C3  H6  03 
Dextrose     Lactic  acid 

In  the  same  way  lactic  acid  may  be  produced  by  bacteria  from 
levulose. 

Examples  of  lactic  acid  formation  are  furnished  by  the  strepto- 
coccus lacticus,  and  B.  lactis  aerogenes.  In  the  case  of  the  former, 
the  fermentation  may  indeed  proceed  'by  the  simple  chemical  process 
indicated  in  the  formulae,  since  the  action  of  the  bacillus  is  entirely 
unaccompanied  by  the  evolution  of  gas. 

Numerous  other  bacteria  produce  large  amounts  of  lactic  acid 
from  lactose,  possibly  by  chemical  processes  less  simply  formulated. 
Among  these  are  bacilli  of  the  colon  group,  B.  prodigiosus,  B.  proteus 
vulgaris,  and  many  others.  Although  lactic  acid  is  usually  the  chief 
product  in  the  bacterial  fermentation  of  the  simpler  carbohydrates, 
acetic,  formic,  and  butyric  acids  may  often  be  found  as  by-products 
in  variable  amounts.16 

Oxy  doses  (Oxydizing  Enzymes).  —  The  most  common  example  of 
oxidation  by  means  of  bacterial  ferments  is  the  production  of  acetic 
acid  from  weak  solutions  of  ethyl  alcohol.  This  process,  which  is 
the  basis  of  vinegar  production,  is  universally  carried  out  by  bac- 
terial ferments.  While  possessed  to  some  extent  by  a  considerable 
number  of  microorganisms,  acetic  acid  formation  is  a  function  pre- 
eminently of  the  bacterial  groups  described  by  Hansen,  including 
"Bacterium  aceti"  and  "Bacterium  pasteurianum.  "  To  these  two 
original  groups,  a  number  of  others  have  since  been  added. 

The  organisms  are  short,  plump  bacilli,  with  a  tendency  to  chain- 
formation,  and  occasionally  showing  characteristically  swollen 
centers  and  many  irregiilar  involution  forms.  In  the  production 
of  vinegar,  as  generally  practiced  by  the  farmer  witli  cider  or  wine, 
these  bacteria  accumulate  on  the  surface  of  the  fluid  as  a  pellicle 


™Buchner  und  Meisenheimer,  Ber.  d.-Deut.  chem.  Gesellsch.,  xxxvi,  1903. 


58  BIOLOGY  AND  TECHNIQUE 

or  scum  which  is  popularly  known  as  the  "mother  of  vinegar." 
Destruction  of  these  bacteria  by  disinfectants  or  by  sterilization 
with  heat  promptly  arrests  the  process  of  vinegar  formation. 
Chemically,  the  conversion  of  the  alcohol  consists  in  a  double  oxida- 
tion through  ethyl  aldehyde  into  acetic  acid  as  shown  in  the  follow- 
ing formulae : 

1.  C2H5  (OH)  +  0  =  CH3  (COH) 
Alcohol  Ethyl  aldehyde 

2.  CH3  (COH)  +  O  =  CH3  (COOH) 
Acetic  acid 

Alcoholic  Fermentation  (Zymase). — The  formation  of  alcohol  as 
an  end  product  of  fermentation  is  of  great  importance  in  a  number 
of  industries,  primarily  in  the  production  of  wine  and  beer. 
While  accomplished  by  a  number  of  bacteria,  this  form  of  fermen- 
tation is  carried  out  chiefly  by  the  yeasts. 

Expressed  in  formulae  the  simplest  varieties  of  alcoholic  fermen- 
tation, from  mono-  and  disaccharids,  may  be  represented  as  follows : 

C6H1206  ==  2C2H5  (OH)  +  2C02 
Dextrose         Ethyl  alcohol 
or 

C12H2Ai  +  H2  0  =  4C2H5(OH)  +  4C02 
Saccharose  Ethyl  alcohol 

In  all  cases  the  process  may  not  be  so  simple  as  indicated  by  the 
equations,  since  by-products,  such  as  higher  alcohols,  glycerin,  suc- 
cinic  and  acetic  acids,  may  often  be  found  in  small  traces  among 
the  end-products  of  such  fermentations.  The  conditions  which  favor 
alcoholic  fermentation  by  the  yeasts  are  extremely  important,  since, 
upon  observance  of  these,  depends  much  of  the  uniformity  of  result 
which  is  so  desirable  in  the  industries  mentioned  above.  The  opti- 
mum concentration  of  sugar  for  the  production  of  the  highest  quan- 
tity of  alcohol  is  at  or  about  25  per  cent.  The  temperature  favoring 
the  process  ranges  about  30°  C.  Under  such  conditions  fermenta- 
tion may  continue  until  the  alcohol  forms  almost  a  20-per-cent 
solution.  Most  of  the  fermentations  important  in  the  wine,  beer, 
and  spirit  industries,  take  place  under  anaerobic  conditions,  since 
the  carbon  dioxide  which  is  formed  soon  shuts  out  any  excess  of  air. 


THE   BIOLOGICAL  ACTIVITIES  OF   BACTERIA  50 

In  the  industrial  employment  of  yeasts  for  fermentative  pur- 
poses, it  is  necessary  to  work  with  specific  strains,  and  in  scien- 
tifically conducted  vineyards,  breweries,  and  distilleries  the  study 
and  pure  cultivation  of  the  yeasts  form  no  unimportant  part  of 
the  work.  Certain,  races  of  yeasts  are  more  uniform  in  their  fermen- 
tative powers  than  others,  and  the  by-products  formed  by  some 
races  differ  sufficiently  from  those  of  other  races  to  cause  material 
differences  in  the  resulting  substances.  In  the  wine  industries, 
the  yeasts  differ  much  from  one  another  according  to  climatic  and 
other  environmental  conditions.  In  vineyards,  natural  inoculation 
of  the  grapes  occurs  by  transportation  of  the  yeast  from  the  soil 
to  the  surface  of  the  grapes  by  wasps,  bees,  or  other  insects,  through 
whose  alimentary  canals  the  microorganisms  pass  uninjured.  In  the 
autumn  the  yeast  is  returned  to  the  soil  by  falling  berries  and 
remains  alive  in  the  upper  layers  of  the  ground  throughout  the 
winter  months.  In  actual  practice  this  natural  yeast  inoculation  is 
not  depended  upon,  but  pure  cultures  of  artificially  cultivated  yeasts 
are  employed  for  inoculation.  In  some  of  the  wine-growing  coun- 
tries these  are  supplied  by  special  government  experiment  stations. 

Denitrifying  Bacteria. — Nitrogen  is  most  readily  absorbed  by 
plants  in  the  form  of  nitrates.  These  are  furnished  to  the  soil  chiefly 
by  the  protein  decomposition  induced  by  the  proteolytic  bacterial 
enzymes.  It  is  self-evident,  therefore,  that  any  cleavage  which 
reduces  nitrogenous  matter  beyond  the  stage  of  nitrates,  to  nitrites 
and  ammonia,  detracts  from  the  value  of  the  nitrogen  as  a  food 
stuff  for  plants,  and  the  eventual  setting  free  of  nitrogen  in  the 
elementary  state  renders  it  entirely  valueless  for  any  but  the 
leguminous  plants. 

Nevertheless,  this  process  of  nitrogen  waste  or  denitrification 
is  constantly  going  on  in  nature.  In  the  course  of  ordinary  decom- 
position, there  is  a  constant  reduction  of  nitrogenous  matter  to 
nitrites  and  salts  of  ammonia,  actively  taken  part  in  by  a  host  of 
bacteria,  as  many  as  85  out  of  109  investigated  by  Maassen17  being 
found  to  possess  this  power.  This,  however,  is  not  nearly  so  harmful 
a  source  of  nitrogen  waste  as  the  process  technically  spoken  of  as 
true  denitrification,  in  which  nitrates  are  reduced,  through  nitric 
and  nitrous  oxides,  to  elementary  nitrogen. 

This  phenomenon,  more  widely  spread  among  bacteria  than  at 


"  Maassen,  Arb.  a.  d.  kais.  Gesundheitsamt,  1,  xxviii,  1901. 


60  BIOLOGY  AND  TECHNIQUE 

first  believed,  depends  essentially  upon  simple  oxygen  extraction 
from  the  nitrates  by  the  bacteria,  and  for  this  reason  goes  on  most 
actively  when  the  supply  of  atmospheric  oxygen  is  low.  The  first 
bacteria  described  as  possessing  this  power  of  denitrification  were 
the  so-called  B.  denitrificans  I  and  II,  the  first  an  obligatory 
anaerobe,  the  other  a  facultative  aerobe.  Since  then  numerous  other 
bacteria,  among  them  B.  coli  and  B.  pyocyaneus,  have  been  shown 
to  exhibit  similar  activities.  It  is  important  agriculturally,  there- 
fore, to  know  that  many  species  which  are  able  to  utilize  atmospheric 
oxygen  when  supplied  with  it,  will  get  their  oxygen  by  the  reduction 
of  nitrates  and  nitrites  when  free  oxygen  is  withheld.  It  is  thus 
clear  that  a  loss  of  nitrogen  is  much  more  apt  to  proceed  rapidly 
in  manure  heaps  which  are  piled  high  and  poorly  aerated.  There 
are  other  factors,  however,  in  regard  to  the  physiology  of  these 
microorganisms,  which  must  be  considered  for  practical  purposes. 

In  order  that  these  bacteria  may  develop  their  denitrifying 
powers  to  the  best  advantage,  it  is  necessary  to  supply  them  with 
some  carbon  compound  which  is  easily  absorbed  by  them.  This,  in 
decomposing  material,  is  furnished  by  the  products  of  the  carbohy- 
drate cleavage  going  on  side  by  side  with  the  proteolytic  processes. 
It  is  still  more  or  less  an  open  question  whether  the  facilitation  of 
denitrification  brought  about  in  manure  heaps  by  the  presence  of 
hay  and  straw  is  due  to  the  carbon  furnished  by  these  materials, 
or  whether  it  is  due  to  the  fact  that  bacilli  of  this  group  are  apt 
to  adhere  to  the  straw  which  acts  in  that  case  as  a  means  of 
inoculation. 

The  actual  danger  of  -nitrogen  depletion  of  the  soil  by  denitri- 
fying processes  is  probably  much  less  threatening  than  was  formerly 
supposed ;  for,  in  the  first  place,  the  conditions  for  complete  denitri- 
fication are  much  more  perfect  in  the  experiment  than  they  ever 
can  be  in  nature,  and  the  nitrifying  processes  going  on  side  by 
side  with  denitrification  make  up  for  much  of  the  loss  sustained. 

ANABOLIC  OR  SYNTHETIC  ACTIVITIES  OP  BACTERIA 

Nitrogen  Fixation  by  Bacteria.— The  constant  withdrawal  of 
nitrogenous  substances  from  the  soil  by  innumerable  plants  would 
soon  lead  to  total  depletion  were  it  not  for  certain  forces  continually 
at  work  replenishing  the  supply  out  of  the  large  store  of  free 
nitrogen  in  the  atmosphere.  This  important  function  of  returning 


THE  BIOLOGICAL  ACTIVITIES  OF  BACTERIA  61 

nitrogen  to  the  soil  in  suitable  form  for  consumption  by  the  plants 
is  performed  largely  by  bacteria. 

It  is  well  known  that  specimens  of  agricultural  soil  when  allowed 
to  stand  for  any  length  of  time  without  further  interference  will 
increase  in  nitrogenous  content,  but  that  similar  specimens,  if 
sterilized,  will  show  no  such  increase.18  The  obvious  conclusion  to 
be  drawn  from  this  phenomenon  is  that  some  living  factor  in  the 
unsterilized  soil  has  aided  in  increasing  the  nitrogen  supply.  Light 
was  thrown  upon  this  problem  when  Winogradsky,19  in  1893,  dis- 
covered a  microorganism  in  soil  which  possessed  the  power  of 
assimilating  large  quantities  of  nitrogen  from  the  air.  This  bac- 
terium, which  he  named  ' '  Clostridium  Pasteurianum,"  is  an  obliga- 
tory anaerobe  which  in  nature  always  occurs  in  symbiosis  with  two 
other  facultatively  anaerobic  microorganisms.  In  symbiosis  with 
these,  it  can  be  cultivated  under  aerobic  conditions  and  thus  grows 
readily  in  the  upper  well-aerated  layers  of  the  soil. 

Although,  until  now,  no  other  bacteria  with  equally  well-devel- 
oped nitrogen-fixing  powers  have  been  discovered,  yet  it  is  more 
than  likely  that  Clostridium  Pasteurianum  is  not  the  only  micro- 
organism endowed  with  this  function.  In  fact,  Penicillium  glaucum 
and  Aspergillus  niger,  two  molds,  and  two  other  bacteria  described 
by  Winogradsky,  have  been  shown  to  possess  this  power  slightly, 
but  in  an  incomparably  less  marked  degree  than  Clostridium  Pas- 
teurianum.20 According  to  the  calculations  of  Sachse,21  unsterilized 
soil  may,  under  experimental  conditions,  gain  as  much  as  25  milli- 
grams of  nitrogen  in  a  season,  a  statement  which  permits  the  cal- 
culation of  a  gain  of  twelve  kilograms  of  nitrogen  per  acre  annually.22 
It  is  very  unlikely,  however,  that  such  gains  actually  occur  in  nature, 
where  nitrogen-fixation  and  nitrogen-loss  usually  occur  side  by  side. 

Agriculturally  of  even  greater  importance  than  the  free  nitrogen- 
fixing  bacteria  of  the  soil  are  the  bacteria  found  in  the  root  tubercles 
of  a  class  of  plants  known  as  "leguminosse. "  It  has  long  been 
known  that  this  class  of  plants,  including  clover,  peas,  beans,  vetch, 
etc*,  not  only  does  not  withdraw  nitrogen  from  the  soil,  but  rather 
tends  to  enrich  it.  Upon  this  knowledge  has  depended  the  well- 

18  Berthelot,  Compt.  rend,   de  la  soc.  de  biol.,  cxvi,  1893. 

19  Winogradsky,  Compt,  rend,  de  la  soc.  de  biol.,  cxvi,  1893,  ibid.,  t.  cxviii,  1894. 

20  Tackc,  Landwirtseh.  Jahresber.,  xviii,  1889. 
slSactec,  "Agr.  Chem.,"  1883. 

-  Pfcffer,  Pfliigers  Physiologic,  p.  395. 


62  BIOLOGY  AND  TECHNIQUE 

known  method  of  alternation  of  crops  employed  by  farmers  the 
world  over.  The  actual  reason  for  the  beneficial  influence  of  the 
leguminosae,  however,  was  not  known  until  1887,  when  Hellriegel 
and  Wilfarth23  succeeded  in  demonstrating  that  the  nitrogen-ac- 
cumulation was  directly  related  to  the  root  tubercles  of  the  plants 
and  to  the  bacteria  contained  within  them. 

These  tubercles,  which  are  extremely  numerous — as  many  as  a 
thousand  sometimes  occurring  upon  one  and  the  same  plant — are 
formed  by  the  infection  of  the  roots  with  bacteria  which  probably 
enter  through  the  delicate  root-hairs.  They  vary  in  size,  are  usually 
situated  near  the  main  root-stem,  and,  in  appearance,  are  not  unlike 
fungus  growths.  Their  development  is  in  many  respects  comparable 
to  the  development  of  inflammatory  granulations  in  animals  after 
infection,  inasmuch  as  the  formation  of  the  tubercle  is  largely  due 
to  a  reactionary  hyperplasia  ol  the  plant  tissues  themselves.  They 
appear  upon  the  seedlings  within  the  first  few  weeks  of  their  growth 
as  small  pink  nodules,  and  enlarge  rapidly  as  the  plant  grows.  At 
the  same  time,  later  in  the  season,  when  the  plants  bear  fruit,  the 
root  tubercles  begin  to  shrink  and  crack.  When  the  crops  are 
harvested,  the  tubercles  with  the  root  remain,  rot  in  the  ground, 
and  re-infect  the  soil. 

Histologically  the  tubercles  are  seen  to  consist  of  large  root  cells 
which  are  densely  crowded  with  microorganisms. 

The  microorganism  itself,  "Bacillus  radicicola,"  was  first  ob- 
served within  the  tubercles  by  Woronin24  in  1866.  The  bacilli  are 
large,  slender,  and  actively  motile  during  the  early  development  of 
the  tubercles,  but  in  the  later  stages  assume  a  number  of  character- 
istic involution  forms,  commonly  spoken  of  as  "  bacteroids. "  They 
become  swollen,  T  and  Y  shaped,  or  branching  and  threadlike.  Their 
isolation  from  the  root  tubercles  usually  presents  little  difficulty, 
since  they  grow  readily  upon  gelatin  and  agar  under  strictly  aerobic 
conditions.  On  the  artificial  media  the  bacillary  form  is  usually  well 
retained,  involution  forms  appearing  only  upon  old  cultures. 

The  classical  experiments  of  Hellriegel  and  Wilfarth  conclusively 
demonstrated  the  important  relation  of  these  tubercle-bacteria  to 
nitrogen  assimilation  by  the  leguminosag. 

These   observers   cultivated  various  members   of  this   group   of 

28  Hellriegel  und  Wilfarth,  Cent.  f.  Bakt.,  1887. 
24  Woronin,  Bot.  Zeit.,  xxiv,  1866. 


N  added  in  seed, 

Harvested 

soil,  and  soil- 

Gain  or 

dry  weight 

N  present 

extract 

loss  of  N 

(  (a)   38.919 

.998 

.022 

+.975 

{   (b)   33.755 

.981 

.023 

+.958 

J   (c)     0.989 

.016 

.020 

—.004 

(  (d)     0.828 

.011 

.022 

—.009 

THE   BIOLOGICAL   ACTIVITIES   OF   BACTERIA  63 

plants  upon  nitrogen-free  soil — sand — and  prevented  the  formation 
of  root  tubercles  in  some,  by  sterilization  of  the  sand,  while  in  others 
they  encouraged  tubercle  formation  by  inoculation.  An  example  of 
their  results  may  be  given  as  follows  :25 

Lupiiius  luteus  was  cultivated  upon  sterilized  sand.  Some  of  the 
pots  were  inoculated  with  B.  radicicola,  others  were  kept  sterile. 
Comparative  analyses  were  made  of  the  plants  grown  in  the  different 
pots  with  the  following  striking  result : 


Root  tubercles  present. 
No   root   tubercles.  . 


The  great  importance  of  this  process  in  agriculture  is  demon- 
strated, furthermore,  by  a  comparison  made  by  the  same  observers 
between  a  legume,  the  pea,  and  one  of  the  common  nitrogen-con- 
suming crops,  oats.26  Exactly  what  the  process  is  by  which  the 
bacteria  supply  nitrogen  to  the  plant  is  as  yet  uncertain.  Although 
the  degenerating  bacteroids  in  old  nodules  are  bodily  absorbed  by 
the  plant,  this  can  not  be  conceived  as  the  only  method  of  supply, 
since  the  total  nitrogen  gain  many  times  exceeds  the  total  weight 
of  bacteria  in  the  nodules.  It  is  probable  that  the  microorganisms 
during  life  take  up  atmospheric  nitrogen  and  secrete  a  nitrogenous 
substance  which  is  absorbed  by  the  plant  cells. 

Although  formerly  the  relationship  between  plant  and  bacterium 
was  regarded  as  one  of  symbiosis  and  of  mutual  benefit,  the  opinions 
as  to  this  subject  show  wide  divergence.  While,  according  to  some 
authors,  the  entrance  of  the  bacteria  into  the  plants  is  regarded 
as  a  true  infection  against  which  the  plant  offers  at  first  a  determined 
opposition  as  evidenced  by  tissue  reactions,  other  observers,  notably 

~*Pfeffer,  "Planzenphysiologie,"  Leipzig,  1897. 

2«Hellriegel  und  Wilfarth,  Zeit.  d.  Ver.  f.  d.  Rubenzucker  Industrie,  1888. 
Quoted  from  Fischer,  "Vorles.  iiber  die  Bakt.,"  Jena,  1903. 

Nitrogen  contents  Nitrogen  contents 

of  seed  and  soil.  of  crop.  Gain  or  loss. 

Oats  0.027  gram  0.007  gram  —.020 

Peas  0.038      "  0.459      "  +.421 


64  BIOLOGY  AND  TECHNIQUE 

A.  Fischer,  regard  the  plant  as  a  parasite  upon  the  bacteria,  in  that 
it  derives  the  sole  benefit  from  the  relationship  and  eventually  bodily 
consumes  its  host. 

Nitrifying  Bacteria. — A  process  diametrically  opposed  in  its 
chemistry  to  denitrification  and  reduction  is  that  which  brings  about 
an  oxidation  of  ammonia  to  nitrites  and  nitrates.  The  actual  in- 
crease of  nitrates  in  soil  allowed  to  stand  for  any  length  of  time 
and  examined  from  time  to  time  has  been  a  well-established  fact 
for  many  years ;  but  it  was  believed  until  a  comparatively  short  time 
ago  that  this  increase  was  due  to  a  simple  chemical  oxidation  of 
ammonia  by  atmospheric  oxygen.  The  dependence  of  nitrification 
upon  the  presence  of  living  organisms  was  finally  proved  by  Muntz 
and  Schlossing27  in  1887,  who  demonstrated  that  nitrification  was 
abruptly  stopped  when  the  soil  was  sterilized  by  heat  or  antiseptics. 
It  remained,  however,  to  isolate  and  identify  the  organisms  which 
brought  about  this  ammonia  oxidation.  This  last  step  in  our  knowl- 
edge of  nitrification  was  taken  in  1890,  by  Winogradsky.  Wino- 
gradsky28  found  that  the  failures  experienced  by  others  who  had 
attempted  to  isolate  nitrifying  bacteria,  were  due  to  the  fact  that 
they  had  used  the  common  culture  media  largely  made  up  of  organic 
substances.  By  using  culture  media  containing  no  organic  matter, 
Winogradsky  succeeded  in  isolating  free  from  the  soil,  bacteria 
which  have  since  that  time  been  confirmed  as  being  the  causative 
factors  in  nitrification.  During  his  first  experiments  this  author 
observed  that  in  some  of  his  cultures  the  oxidation  of  ammonia  went 
only  as  far  as  the  stage  of  nitrite  formation,  while  in  others  complete 
oxidation  to  nitrates  took  place.  Following  the  clews  indicated  by 
this  discrepancy,  he  finally  succeeded  in  demonstrating  that  nitrifica- 
tion is  a  double  process  in  which  two  entirely  different  varieties  of 
microorganisms  take  part,  the  one  capable  of  oxidizing  ammonia  to 
nitrites,  the  other  continuing  the  process  and  converting  the  nitrites 
to  nitrates.  The  nitrite-forming  bacteria  discovered  by  Winograd- 
sky, and  named  Nitromonas  or  Nitrosomonas,  are  easily  cultivated 
upon  aqueous  solutions  containing  ammonia,  potassium  sulphate, 
and  magnesium  carbonate.  According  to  their  discoverer  they 
develop  in  this  medium  within  a  week  as  a  gelatinous  sediment, 
After  further  growth  this  sediment  seems  to  break  up  and  the 


27  Munis  und  Schlossing,  Cornpt.  rend,  de  1'acad.  des  sciences,  1887. 

28  Winogradsky,  Ann.  Past.  Inst.,  iv  and  v,  1890,  ]891. 


THE   BIOLOGICAL  ACTIVITIES  OF  BACTERIA  65 

bacteria  appear  as  oval  bodies,  which  swim  actively  about  and 
develop  flagella  at  one  end.  Upon  the  solid  media  in  ordinary  use 
they  can  not  be  cultivated.  Special  solid  media  suitable  for  their 
cultivation  and  composed  of  silicic  acid  and  inorganic  salts  have 
been  described  by  Winogradsky  and  by  Omeliansky.29 

Other  nitrite-forming  bacteria  have  since  been  described  by 
various  observers,  all  of  them  more  or  less  limited  to  definite  locali- 
ties. Some  of  these  are  similar  to  nitrosomonas  in  that  they  exhibit 
the  flagellated,  actively  motile  stage.  In  others,  this  stage  is  absent. 

The  nitrite-forming  bacteria,  apart  from  their  great  agricultural 
importance,  claim  our  attention  because  of  their  unique  position  in 
relation  to  the  animal  and  vegetable  kingdoms.  Extremely  sensitive 
to  the  presence  of  organic  compounds,  they  are  able  to  grow  and 
develop  only  upon  media  containing  nothing  but  inorganic  material ; 
and  this  entirely  without  the  aid  of  any  substances  comparable  to 
the  chlorophyll  of  the  green  plants.  The  source  of  energy  from 
which  this  particular  class  of  bacteria  derive  the  power  of  building 
up  organic  compounds  from  simple  substances  is  to  some  extent  a 
mystery.  The  carbon  which  they  unquestionably  require  for  the 
building  up  of  organic  material  may  be,  as  Winogradsky  believed, 
derived  to  a  certain  extent  from  ammonium  carbonate.  But  it  is 
also  quite  certain  that  they  are  capable  of  utilizing  directly  atmos- 
pheric C02.  In  the  absence  of  chlorophyll  or  of  any  highly  or- 
ganized chemical  compound,  it  seems  likely  that  the  energy  neces- 
sary for  the  utilization  of  the  carbon  obtained  in  this  simple  form 
is  derived  from  the  oxidation  of  ammonia  during  the  process  of 
nitrification. 

The  conversion  of  nitrites  into  nitrates  is  carried  on  by  other 
species  of  bacteria  also  discovered  by  Winogradsky.  These  bacteria 
are  much  more  generally  distributed  than  nitrosomonas  and  prob- 
ably include  a  number  of  varieties.  The  organism  described  by 
Winogradsky  is  an  extremely  small  bacillus  with  pointed  ends.  Cap- 
sules have  occasionally  been  demonstrated.  It  may  be  cultivated 
upon  aqueous  solutions  containing: 

Sod.  nitrite   1     per  cent. 

Potass,   phosphate    05    "      ' ' 

Magnesium   sulphate    08    < l       ' ' 

Sodium    carbonate :  .  .    .1      ' '       ' ' 

Ferrous  sulphate 04    "       " 

29  Omeliansky,  Cent.  f.  Bakt.,  II,  5,  1899. 


66  BIOLOGY  AND  TECHNIQUE 

The  development  of  the  organism  is  slow  and  sparse,  and  is  directly 
inhibited  by  the  presence  of  organic  matter.  It  is  strongly  inhibited 
by  the  presence  of  ammonia. 

The  Liberation  of  Energy  by  Bacteria. — Like  all  other  living 
beings,  bacteria  in  their  metabolic  processes  liberate  energy.  It  has 
been  shown  by  several  observers  that  slight  quantities  of  heat  are 
given  off  from  actively  growing  cultures.  The  functions,  further- 
more, of  reproduction,  motility,  and  enzyme  formation  may  be  looked 
upon  as  forms  of  energy  liberation.  In  addition  to  this,  certain 
bacteria  have  been  observed  which  may  liberate  energy  in  the  form 
of  light. 

Light  Production  by  Bacteria.- -The  production  of  light  by  bac- 
teria is  a  power  possessed  chiefly  by  certain  species  inhabiting  salt 
water.  Thus,  much  of  the  phosphorescence  observed  at  sea,  though 
more  frequently  due  to  Medusa  and  other  invertebrate  animals,  is 
caused  by  these  bacteria.  Numerous  species  which  produce  this 
phenomenon  have  been  isolated,  too  many,  and  too  unimportant, 
to  be  individually  described.  All  of  them  are  aerobes  and  require 
highly  complex  food  stuffs.  They  are  closely  allied  to  the  putre- 
factive bacteria,  and  in  the  sea  are  usually  found  upon  rotting 
animal  matter.30  The  production  of  light  seems  directly  dependent 
upon  the  free  access  of  oxygen,  since  no  light  appears  under 
anaerobic  conditions.  Their  luminous  quality,  moreover,  is  not  a 
true  phosphorescence,  in  that  it  does  not  depend  upon  previous 
illumination  and  develops  as  well  in  cultures  kept  in  the  dark  as 
in  those  which  have  been  exposed  to  light.31 

The  Formation  of  Pigment  by  Bacteria  (Chromobacteria) .— A 
large  number  of  bacteria,  when  cultivated  upon  suitable  media,  give 
rise  to  characteristic  colors  which  are  valuable  as  marks  of  differen- 
tiation. For  each  species,  the  color  is  usually  constant,  depending, 
to  a  certain  extent,  upon  the  conditions  of  cultivation.  In  only  a 
few  of  the  pigmented  bacteria  is  the  pigment  contained  within  the 
cell  body,  and  in  only  one  variety,  the  sulphur  bacteria,  does  the 
pigment  appear  to  hold  any  distinct  relationship  to  nutrition.  In 
most  cases,  the  coloring  matter  is  found  to  be  deposited  in  small 
intercellular  granules  or  globules.  The  absence  of  any  relationship 
of  the  pigment  to  sunlight,  as  is  the  case  with  the  chlorophyll  of 

^Pfliigcr's  Arch.  f.  Phys.,  xi,  1875. 
31  Fischer,  Cent.  f.  Bakt.,  iii,  1888. 


THE  BIOLOGICAL  ACTIVITIES  OF  BACTERIA  67 

the  green  plants,  is  indicated  by  the  fact  that  most  of  the  chrorno- 
bacteria  thrive  and  produce  pigment  equally  well  in  the  dark  as 
they  do  in  the  presence  of  light.  Among  the  most  common  of  the 
pigment  bacteria  met  with  in  bacteriological  work  are  Staphylo- 
coccus  pyogenes  aureus,  Bacillus  pyocyaneus,  Bacillus  prodigiosus, 
and  some  of  the  green  fluorescent  bacteria  frequently  found  in  feces. 

The  chemical  nature  of  these  pigments  has  been  investigated 
quite  thoroughly  and  it  has  been  shown  that  they  vary  in  com- 
position. Some  of  the  pigments,  like  that  of  Staphylococcus  aureus, 
are  probably  non-protein  and  of  a  fatty  nature.32  They  are  insoluble 
in  water  but  soluble  in  alcohol,  ether,  and  chloroform.  Because  of 
their  probable  composition,  they  have  been  spoken  of  as  "lipo- 
chromes. "  Other  pigments,  like  the  pyocyanin,  which  lends  the 
green  color  to  cultures  of  Bacillus  pyocyaneus,  are  water  soluble 
and  are  probably  of  protein  composition.  Pyocyanin  may  be 
crystallized  out  of  aqueous  solution  in  the  form  of  fine  needles.  The 
crystals  may  be  redissolved  in  chloroform.  Aqueous  solutions  retain 
their  color.  Solutions  in  choloroform,  however,  are  changed  grad- 
ually to  yellow. 

The  power  of  pigment  production  of  various  bacteria  depends  in 
each  case  upon  cultural  conditions.  In  most  cases,  this  simply  sig- 
nifies that  pigment  is  produced  only  when  the  microorganism,  finding 
the  most  favorable  environmental  conditions,  is  enabled  to  develop 
all  its  functions  to  their  fullest  extent.  Thus,  a  too  high  acidity  or 
alkalinity  of  the  culture  medium  may  inhibit  pigment  formation. 
Oxygen  is  necessary  for  the  production  of  color  in  some  bacteria, 
since  the  bacteria  themselves  often  produce  the  pigment  only  as  a 
leuko-body  which  is  then  oxidized  into  the  pigment  proper.  A 
notable  example  of  this  is  the  pigment  of  B.  pyocyaneus.  In  other 
cases,  temperature  plays  an  important  role  in  influencing  color  pro- 
duction. Thus,  Bacillus  prodigiosus  does  not  produce  its  pigment 
when  growing  in  the  incubator.  By  persistent  cultivation  in  an 
unfavorable  environment,  colored  cultures  may  lose  their  power  of 
pigment  production. 

Sulphur  Bacteria. — Wherever  the  decomposition  of  organic  mat- 
ter gives  rise  to  the  formation  of  H2S,  in  cess-pools,  in  ditches,  at 
the  bottom  of  the  sea,  and  in  stagnant  ponds,  there  is  found  a 
curiously  interesting  group  of  microorganisms,  the  so-called  sulphur 


82  Schroetcr,  Cent,  f .  Bakt.,  xviii,  1895. 


68  BIOLOGY  AND  TECHNIQUE 

or  thiobacteria.  Red,  purple,  and  colorless,  these  bacteria  all  possess 
the  power  of  utilizing  sulphuretted  hydrogen  and  by  its  oxidation 
into  free  sulphur  obtain  the  energy  necessary  for  their  metabolic 
processes.  The  colorless  sulphur  bacteria,  the  Beggiatoa  and 
Thiothrices,  usually  appear  as  threads  or  chains  which,  in  media 
containing  sufficient  H2S,  are  usually  well-stocked  with  minute 
globules  of  sulphur.  If  found  upon  decomposing  organic  matter, 
they  often  cover  this  as  a  grayish  mold-like  layer.  The  red  sulphur 
bacteria,  of  which  numerous  species  have  been  described  by  Wino- 
gradsky,  may  appear  as  actively  motile  spirilla  (thiospirillum)  or 
as  short,  thick  bacillary  forms. 

The  physiology  of  all  the  sulphur  bacteria,  and  especially  of  the 
colored  varieties,  is  of  the  greatest  interest  in  that  these  micro- 
organisms are  among  the  few  members  of  the  bacterial  group  which 
behave  metabolically  like  the  green  plants.  The  higher  organic 
substances  play  little  or  no  part  in  the  nutrition  of  these  micro- 
organisms. Strictly  aerobic,  the  colorless  thiobacteria  are  inde- 
pendent of  sunlight,  while  the  red  and  purple  varieties  exhibit  their 
physiological  dependence  upon  light  by  accumulating  under  natural 
conditions  in  well-lighted  spots.  Both  varieties  possess  equally  the 
power  of  oxidizing  sulphuretted  hydrogen  as  a  source  of  energy. 
The  sulphur  is  then  stored  as  elemental  sulphur  within  the  bacterial 
body  and  when  a  lack  of  food  stuffs  sets  in,  the  store  of  sulphur 
can  be  further  oxidized  into  sulphurous  or  sulphuric  anhydrides. 
With  this  sole  source  of  energy,  these  bacteria  are  capable  of  flourish- 
ing aerobically,  while  an  absence  of  H2S,  even  in  the  presence  of 
organic  food  stuffs,  leads  to  a  rapid  disappearance  of  their  sulphur 
contents  and  an  inability  to  develop. 

In  the  case  of  the  colored  thiobacteria,  the  red  pigment  appears 
to  fulfil,  to  some  extent,  a  function  comparable  to  that  of  the 
chlorophyll  of  the  green  plants. 

Engelmann,33  who  has  studied  this  pigment  spectroscopically,  has 
found  that  besides  absorbing  the  red  spectral  rays  there  is  an  ab- 
sorption of  rays  on  the  ultra-red  end  of  the  spectrum.  The  absorp- 
tion of  the  red  rays  between  the  lines  B  and  C  of  the  spectrum, 
and  of  violet  rays  at  the  line  F,  is  the  same  as  that  of  the  absorption 
spectrum  of  chlorophyll,  and  it  is  in  the  zone  of  these  rays  that  the 
physiological  effects  of  cholorophyll  are  most  active.  In  addition 


**  Engelmann,  Bot.  Zeit.,  1888. 


THE  BIOLOGICAL  ACTIVITIES  OF  BACTERIA  69 

to  these  absorption  bands,  the  bacteriopurpurin  of  the  red  sulphur 
bacteria  shows  absorption  of  the  invisible  ultra  red  rays  of  the 
spectrum. 

Engelmann,  with  a  microspectroscope,  projected  a  spectrum  into 
a  microscopic  field  in  which  green  algae  or,  in  the  case  under  discus- 
sion, red  sulphur  bacteria  had  been  placed.  Other  sources  of  light 
were,  of  course,  excluded.  By  adding  emulsions  of  strictly  aerobic 
bacteria  to  such  preparations,  an  accumulation  of  microorganisms 
was  observed  at  those  points  in  the  spectrum  at  which  most  oxygen 
was  liberated.  In  the  case  both  of  chlorophyll  and  of  the  red  sul- 
phur bacteria  such  areas  of  bacterial  accumulation  (in  oxygen 
liberation)  occurred  in  the  zones  of  the  absorption  bands  mentioned 
above. 

THE  SO-CALLED  ' '  BACTERIOPHAGE ' '  PHENOMENON 

During  the  last  few  years  a  phenomenon  has  been  observed  with 
bacteria  which  is  likely  to  be  of  fundamental  importance  in  the 
biological  and  immunological  study  of  bacteriology. 

The  first  observation  upon  this  curious  phenomenon  was  pub- 
lished by  Twort  in  1915.3*  Twort  was  engaged  in  a  search  for  a 
non-pathogenic  filtrable  virus  on  the  very  logical  assumption  that 
the  few  pathogenic  filtrable  virus  which  had  been  associated  with 
a  number  of  diseases  probably  represented  isolated  members  of  a 
large  kingdom  of  microorganisms.  His  cultural  and  animal  studies 
proving  negative  by  the  ordinary  methods,  he  carried  out  experi- 
ments with  glycerinated  calf  vaccine  which  he  cultured  on  agar 
tubes.  After  variable  periods  of  incubation  such  agar  slants  would 
exhibit  watery  areas  and  in  those  cultures  in  which  micrococci  grew, 
it  was  found  that  some  of  the  colonies  could  not  be  sub-cultured, 
but  if  preserved,  the  originally  normal  culture  would  become  glassy 
and  transparent.  Smears  made  from  these  glassy  areas  showed  red- 
dish staining  granules  by  Giemsa.  Plating  of  the  condensation  water 
of  such  slants  resulted  in  colonies  which  usually  after  preservation 
became  transparent,  starting  unlike  most  degenerative  processes, 
from  the  edge  of  the  colony.  Pure  cultures  of  pneumococci  and 
other  microorganisms  when  touched  with  small  portions  of  the  glassy 
colonies  would  become  transparent  at  the  point  touched  and  this 

84  Twort,  Lancet,  December  4th,  1915. 


70  BIOLOGY  AND  TECHNIQUE 

would  gradually  spread  over  the  whole  group.  Such  action  was 
complete  and  rapid  only  with  vigorously  growing  young  cultures, 
but  had  little  effect  on  dead  cultures.  The  action  took  place  best 
under  aerobic  conditions.  If  the  transparent  material  was  diluted 
and  filtered  and  then  added  to  an  agar  slant  of  similar  organisms, 
growth  was  prevented.  The  transparent  material  did  not  grow  on 
any  medium  and  was  destroyed  in  its  activity  at  60°.  Its  action 
on  staphylococci  grown  from  boils  was  very  slight,  and  there  was 
no  action  of  the  substance  upon  B.  coli,  streptococci,  tubercle  bacilli 
or  yeasts.  The  nature  of  the  material  was  left  undecided  by  Twort. 
It  was  supposed  that  it  might  be  a  parasitic  organism  which  de- 
stroyed the  bacteria,  perhaps  an  amoeba,  but  he  thought  that  it 
was  probably  a  substance  derived  from  the  bacteria  themselves, 
since  it  occasionally  reappeared  in  glassy  cultures  months  after  sub- 
culture. Twort  investigated  this  peculiar  occurrence  not  only  with 
vaccine  virus,  but  also  with  Gram-negative  bacilli  obtained  from 
the  intestines  of  a  dog  suffering  from  acute  distemper  and  some 
organisms  derived  from  infantile  diarrhea. 

In  1917  D'Herelle35  began  the  publication  of  a  series  of  notes 
upon  a  phenomenon  unmistakably  identical  with  that  of  Twort. 
When  he  introduced  two  or  three  drops  of  a  dysentery  stool  into 
about  20  c.c.  of  broth  and  filtered  this  through  a  Chamberland 
Candle  and  introduced  a  trace  of  this  filtrate  into  a  young  Shiga 
bacillus  culture,  it  caused  a  clearing  up  of  the  culture  within  a 
few  hours,  transplants  remaining  sterile.  A  trace  of  this  dissolved 
culture  placed  into  another  young  broth  culture  of  dysentery 
brought  the  same  result,  and  in  this  way  he  made  935  successive 
passages  of  the  lytic  principle.  His  technique  in  detail  was  as  fol- 
lows :  He  made  an  emulsion  of  fecal  material  and  filtered  it  through 
a  filter  candle.  With  a  pipette  he  then  transferred  1  c.c.  of  this 
filtrate  into  a  small  tube  and  inoculated  this  material  with  a  drop 
of  a  young  dysentery  or  typhoid  culture.  This  tube  is  held  for 
from  12  to  24  hours  at  a  temperature  of  22°,  and  is  then  placed 
for  2  or  3  hours  at  37°.  If  the  stool  filtrate  contains  a  lytic  prin- 
ciple, the  inoculated  tube  will  be  transparent  at  the  end  of  this 
time,  and  the  lytic  principle  can  be  transmitted  in  series  from  this 
tube.  He  assumed  from  his  ability  to  keep  this  lytic  activity  going 


33  D  'Herelle,  Compt.  Kend,  de  la  Soc.  Biol.,  81,  1918,  1160,  82,  1919,  1237,  83, 
1920,  52,  97  and  247. 


THE   BIOLOGICAL  ACTIVITIES   OF   BACTERIA  71 

through  many  generations  that  he  was  dealing  with  a  living  ultra- 
microscopic  organism  which  fed  upon"  the  living  bacteria  and,  there- 
fore, spoke  of  it  as  a  "  bacteriophage. ' ' 

A  curious  and  important  phase  of  the  work  was  that  this  lytic 
principle  was  to  some  extent  specific  in  that  it  acted  only  upon 
a  single  or  a  few  closely  related  microorganisms. 

In  subsequent  notes  D'Herelle  reported  that  he  had  produced 
such  bacteriophage  lytic  principles  against  dysentery  bacilli  of 
Shiga  and  Flexner  and  "Y"  type,  against  typhoid,  paratyphoid 
"A"  and  "B,"  against  enteritidis,  hog  cholera,  coli,  prodigiosus  and 
some  other  Gram-negative  bacilli.  Without  wishing  to  detail  all 
his  investigations,  the  principles  may  be  summarized  as  follows: 
In  all  cases  the  lytic  principle  was  filtrable  and  could  be  carried 
on  from  generation  to  generation  as  above  described.  Young  ac- 
tively growing  cultures  were  necessary  to  transmit  the  lytic  activity 
in  series;  it  could  not  be  transmitted  by  dead  bacteria.  The  lytic 
principle  and  the  bacilli  were  not  killed  by  the  same  temperature. 
Whereas  the  Shiga  bacillus  and  other  organisms  worked  with  were 
destroyed  at  or  about  50°  C.,  the  lytic  principle  resisted  tempera- 
tures up  to  65°.  The  lytic  principle  isolated  from  the  feces  might 
at  first  be  feeble,  but  was  considerably  augmented  in  potency  by 
cultures  in  series.  Certain  cultures  were  from  the  beginning  en- 
dowed with  bacteriolytic  power  against  two  or  more  different  species 
and  preserved  this  action  against  these  species  for  a  long  time  if 
cultivated  only  on  one  of  them.  For  example,  he  isolated  an  active 
principle  against  the  Shiga  and  typhoid  bacillus  from  the  stool  of 
a  dysentery  convalescent.  After  a  series  of  1,000  passages,  the 
successive  cultures  always  being  carried  along  on  Shiga  emulsions, 
there  was  still  marked  lytic  action  against  typhoid  bacilli.  He  never 
isolated  two  strains  that  were  exactly  alike.  The  intensity  of  action 
differed  and  one  strain  would  from  the  beginning  show  lytic  power 
against  a  number  of  intestinal  bacilli.  Others,  again,  would  be 
active  against  only  a  single  species.  Another,  again,  might  attack 
only  two,  the  Flexner  bacillus  and  paratyphoid  "B." 

D'Herelle  was  quite  definitely  convinced  that  he  was  dealing 
with  an  ultra-microscopic  organism  which  perhaps  was  capable  of 
developing  in  the  intestines  of  man  and  animals  at  the  expense  of 
various  bacteria,  and  could  then  be  cultivated  out  of  the  body  by 
the  method  he  had  developed.  This  was  the  reason  for  the  name 
he  applied  to  those  principles,  "bacteriophage,"  which  implied  that 


72  BIOLOGY  AND  TECHNIQUE 

his  ultra-microscopic  organism  was  a  parasite  upon  bacteria,  adapted 
specifically  in  different  cases  to  various  organisms. 

In  1920  Kabeshima36  studied  the  D'Herelle  phenomenon  and 
interpreted  it  as  being  due  to  ferment  action.  He  believes  that 
D  'Herelle  's  principle  was  a  catalytic  agent  originating  in  the  mucous 
membranes  of  the  intestinal  canal,  which  induced  enzyme  digestion 
of  the  organisms.  In  the  cells  of  bacteria  he  assumes  there  exists 
a  preferment  which  is  activated  by  the  catalyst  and  leads  to 
autolysis  of  the  organisms. 

In  1920,  also,  Bordet  and  Cuica37  attacked  the  same  problem 
from  an  entirely  different  standpoint.  Bordet  injected  guinea  pigs 
intraperitoneally  three  or  four  times  with  B.  coli.  One  or  two  days 
after  the  last  injection,  the  peritoneal  exudate  was  examined  and 
found  to  consist  of  an  exudate  rich  in  leucocytes  in  which  still  a 
few  organisms  were  present.  When  a  few  drops  of  such  an  exudate 
were  added  to  a  normal  B.  coli  culture,  marked  lytic  powers  were 
noticed.  A  small  quantity  of  the  exudate  resulted  in  a  clearing  up 
of  the  normal  broth  culture,  and  a  small  amount  of  this  original 
mixture  would  do  the  same  if  added  to  successive  cultures  of  normal 
Colon  bacilli.  In  other  words,  the  lytic  principle  started  in  the 
peritoneal  exudate  of  guinea  pigs,  was  kept  going  through  successive 
Colon  cultures.  This  active  principle,  like  that  of  D'Herelle,  resisted 
heating  to  from  60°  to  65°,  and  this  sterile  exudate  would  still 
possess  lytic  powers  for  Colon  cultures.  It  may  be  stated  that 
in  Bordet 's  experiments  it  was  found  that  it  was  very  difficult  to 
start  the  principle  in  this  way,  and  that  only  isolated  Colon  cultures 
lent  themselves  to  successful  experimentation.  When  a  few  drops 
of  a  culture  cleared  in  this  way  were  added  to  an  agar  slant  freshly 
inoculated  with  normal  Colon  bacillus,  no  growth  took  place  in  those 
parts  which  had  been  touched  by  the  fluid,  and  colonies  which 
developed  in  or  about  this  area  soon  became  glassy  and  transparent. 
In  the  same  way,  Bordet  often  found  that  dissolved  cultures  were 
not  entirely  sterile  on  sub-culturing.  A  small  number  of  colonies 
developed,  some  of  which  had  an  extremely  irregular  outline.  These 
irregular  colonies  carried  the  lytic  principle  in  subsequent  genera- 
tions. Bordet  interpretes  this  phenomenon  as  the  artificial  produc- 


36  Kabeshima,  Compt.  Rend,  do  la  Soc.  Biol.,  83,  1920. 

37  Bordet  and  Cuica,  Compt.  Bend,  de  la  Soc.  Biol.,  88,  1920, 


THE   BIOLOGICAL   ACTIVITIES  OF   BACTERIA  73 

tion  of  a  variation  in  the  cultures  of  Colon  bacilli  in  which  a  variant 
producing  an  autolytic  enzyme  is  developed,  and  this  property  of 
autolysin  production  is  then  inherited  from  culture  to  culture.  Con- 
ceived in  this  way,  the  lytic  principle  would  be  regarded  as  the 
inheritance  of  an  acquired  characteristic. 

In  our  own  laboratory  we  have  isolated  a  lytic  principle  by  the 
D'Herelle  technique  described  above,  from  the  stool  of  a  typhoid 
convalescent.  It  corresponds  in  most  details  to  the  lytic  principles 
described  by  D'Herelle.  It  is  filtrable,  thermostable,  and  transmit- 
tible  in  series.  It  is  both  inhibitory  and  lytic,  that  is,  a  small  amount 
of  the  original  stool  filtrate  or  any  one  of  the  subsequent  generations 
will  prevent  growth  of  a  heavily  inoculated  tube,  or  dissolve  a  young 
turbid  broth  culture.  This  lytic  principle  is  only  active  against 
closely  allied  species  of  bacteria  of  the  typhoid-paratyphoid  and 
dysentery  groups.  In  the  earlier  experiments  this  lytic  principle 
did  not  appear  active  against  paratyphoid  "A"  or  "B,"  but  when 
retested  recently,  it  was  also  active  against  these  organisms.  It  has 
no  action  against  cholera,  B.  pyocyaneous  or  B.  coli,  or  against 
staphylococcus  or  pneumococcus. 

Unlike  the  lytic  principles  described  by  D'Herelle,  the  one 
isolated  by  us  has  not  increased  in  potency  in  the  course  of  the 
generations  through  which  it  has  been  passed.  It  differs  also  from 
those  described  by  D'Herelle  in  that  it  usually  does  not  sterilize 
the  dissolved  culture  completely.  On  sub-culturing  a  small  number 
of  colonies  develop  which  are  of  two  types:  one  a  round  typical 
typhoid  colony,  the  other  irregular  in  outline.  The  latter  type  was 
first  described  by  Bordet  in  connection  with  the  lytic  principle  that 
he  was  able  to  produce  against  B.  coli  by  intraperitoneal  injections 
of  guinea  pigs,  and  the  bacilli  composing  these  irregular  colonies 
were  shown  by  him  to  be  the  bearer  of  the  lytic  principle. 

The  typical  colonies  which  develop  from  plating  a  dissolved  cul- 
ture, on  restreaking,  give  only  regular  colonies.  If,  however,  one 
of  the  irregular  colonies  are  restreaked  both  regular  and  irregular 
colonies  will  develop.  Daily  restreaking  of  irregular  colonies  have 
failed  to  eliminate  the  typical  normal  colony  in  a  series  of  15 
generations  and  the  irregular  colonies  continued  to  carry  the  lytic 
principle.  If  one  of  these  typical  typhoid  colonies  is  fished  with 
broth,  the  broth  will  become  cloudy  after  incubation.  Broth  fishings 
of  the  irregular  colonies  will,  however,  often  remain  clear  after 
12  to  18  hours  incubation  and  the  lytic  principle  is  then  present  in 


74  BIOLOGY  AND  TECHNIQUE 

the  broth  and  can  be  transmitted  in  series  from  broth  fishings  of 
irregular  colonies  in  the  same  way  as  from  dissolved  cultures. 

If  a  number  of  irregular  colonies  are  obtained  on  a  streak  plate, 
it  will  be  found  that  on  examining  with  the  microscope  or  often 
with  the  naked  eye,  that  there  are  minute  transparent  masses  be- 
tween the  irregular  colonies,  which  are  referred  to  by  most  writers 
as  "appearances."  On  examining  the  irregular  lytic  colonies  under 
the  microscope  it  will  be  found  that  the  lytic  colonies  owe  their 
irregular  shape  to  the  fact  that  their  edges  have  faded  out  into  these 
transparent  appearances.  All  attempt  to  find  a  definite  structure 
in  the  "appearances"  by  different  methods  of  staining  have  failed, 
and  we  have  no  reason  to  believe  that  they  are  living.  In  our 
opinion  the  "appearances"  represent  the  debris  left  when  the  sus- 
ceptible bacilli  in  any  culture  are  dissolved  by  action  of  the  lytic 
principle. 

The  two  most  striking  facts  about  these  lytic  agents  from  what- 
ever source  they  are  derived  are:  (1),  that  a  single  contact  with 
the  lytic  agent  is  sufficient  to  divide  a  normal  culture  into  two  types 
of  organisms,  one  the  resistant  type,  the  other  the  bearer  of  the 
lytic  principle;  (2),  that  the  lytic  principle  could  only  be  transmitted 
in  series  when  young  actively  growing  cultures  were  used.  This 
suggested  that  once  the  dissolving  action  was  started,  the  lytic  agent 
in  subsequent  generations  was  derived  from  the  bacterial  cell  itself. 
Experiments  were  consequently  undertaken  to  isolate  a  lytic  agent 
which  would  start  the  dissolving  process  without  any  interaction 
of  the  living  animal  body. 

The  early  work  of  Twort  in  connection  with  vaccine  virus  sug- 
gested that  tissue  enzymes  might  be  able  to  start  the  process.  In 
consequence  glycerine  extracts  of  intestinal  mucosa  have  been  made 
and  such  extracts  when  added  to  young  turbid  typhoid  or  dysentery 
cultures  in  a  dilution  of  1-10  have  had  a  dissolving  action  on  the 
bacilli  which  is  transmittible  in  series.  Liver  extracts  prepared 
according  to  the  method  of  Turro38  have  given  similar  results. 

Bordet  has  recently  prepared  an  antilytic  serum.39  In  trying 
to  duplicate  his  experiments,  and  testing  the  normal  rabbit  serum 
comparatively  with  the  immune  serum,  it  was  found  that  in  some 
instances  the  normal  rabbit  serum  had  a  dissolving  action  when 


88  Turro,  Compt.  Bend,  de  la  Soc.  Biol.,  February  12th,  1921. 

39  Bordet  and  Ciuca,  Compt.  Eend.  de  la  Soc.  Biol.,  February  5th,  1921. 


THE    BIOLOGICAL   ACTIVITIES  OF   BACTERIA  75 

added  in  a  dilution  of  1-10  to  young  typhoid  broth  cultures.  It 
seems,  therefore,  that  this  power  of  dissolving  bacteria  is  the  prop- 
erty of  many  different  cells  of  the  animal  body  and  is  also  present 
in  certain  instances  in  the  blood  serum.  Whether  the  lytic  agent 
in  the  serum  can  be  definitely  identified  with  serum  protease  or 
lipase  has  not  been  determined. 


CHAPTER  V 

THE  DESTRUCTION  OF  BACTERIA 

GENERAL    CONSIDERATIONS 

No  branch  of  bacteriology  has  been  more  fruitful  in  practical 
application  than  that  which  deals  with  the  factors  which  bring  about 
the  destruction  of  microorganisms.  Upon  the  study  of  this  branch 
has  depended  the  growth  and  the  development  of  modern  surgery. 

The  agents  which  affect  bacteria  injuriously  are  many,  and  are 
both  physical  and  chemical  in  nature. 

When  a  procedure  completely  destroys  bacterial  life  it  is  spoken 
of  as  sterilization  or  disinfection,  the  term  disinfection  being  em- 
ployed more  especially  to  designate  the  use  of  chemical  agents. 
When  the  procedure  destroys  vegetative  forms  only,  leaving  the 
more  resistant  spores  uninjured,  it  is  spoken  of  as  "incomplete 
sterilization."  When  an  agent,  on  the  other  hand,  does  not  actually 
kill  the  microorganisms,  but  merely  inhibits  their  growth  and  mul- 
tiplication, it  is  spoken  of  as  an  antiseptic.  The  term  deodorant 
is  indiscriminately  applied  to  substances  which  mask  or  destroy 
offensive  odors,  and  may  or  may  not  possess  disinfectant  or  antiseptic 
value.  Some  deodorants  act  chemically  on  the  noxious  gases,  de- 
stroying them. 

PHYSICAL    AGENTS    INJURIOUS    TO    BACTERIA 

The  principal  physical  agents  which  may  exert  deleterious  action 
upon  bacteria  are:  drying,  light,  electricity,  and  heat. 

Drying. — Complete  desiccation  eventually  destroys  most  of  the 
pathogenic  bacteria,  yet  great  differences  in  resistance  to  this  con- 
dition are  shown  by  various  microorganisms.  Ficker,1  who  has  made 
a  systematic  study  of  the  influence  of  complete  drying  upon  bacteria, 
concludes  that  the  resistance  of  bacteria  to  desiccation  is  influenced 


1  Ficker,  Zeit.  f .  Hyg.,  xxix,  1896. 

76 


THE   DESTRUCTION   OF  BACTERIA  77 

by  the  age  of  the  culture  investigated,  the  rapidity  with  which  the 
withdrawal  of  moisture  is  accomplished,  and  the  temperature  at 
which  the  process  takes  place.  Microorganisms  like  the  gonococcus 
and  the  Pfeiffcr  bacillus  are  destroyed  by  drying  within  a  few 
hours.  The  cholera  vibrio  dried  upon  a  coverslip  was  found  by 
Koch2  to  be  killed  within  four  hours;  by  Burkholtz,3  to  survive 
about  twenty-four  hours.  The  spore-forms  of  bacteria  are  infinitely 
more  resistant  to  this  influence  than  are  the  vegetative  forms,  though 
they  may  be  destroyed  by  rapid  and  complete  drying  in  a  desiccator. 

It  is  self-evident  that  many  discrepancies  in  the  experimental 
results  of  various  authors  may  depend  upon  the  technique  of  inves- 
tigation, since  the  degree  of  drying  attained  depends  intimately  upon 
the  thickness  and  consistence  of  the  material  investigated,  and  upon 
the  methods  employed  for  desiccation. 

Light. — Direct  sunlight  is  a  powerful  germicide  for  all  bacteria 
except  a  limited  number  of  species  like  the  thio-  or  sulphur  bacteria, 
which  utilize  sunlight  for  their  metabolic  processes  as  do  the  green 
plants. 

Koch*  has  shown  that  exposure  to  sunlight  will  destroy  the  tuber- 
cle bacillus  within  two  hours  or  less,  the  time  depending  upon  the 
thickness  of  the  exposed  layers  and  the  material  surrounding  the 
bacilli.  Confirmatory  researches  have  been  published  by  Mignesco5 
and  others.  The  powerful  disinfecting  influence  of  sunlight  upon 
bacteria  suspended  in  water  has  been  shown  by  Buchner.6  Observa- 
tions in  regard  to  the  influence  of  sunlight  upon  anthrax  spores 
have  been  made  by  Arloing,7  and  similar  observations  upon  a  num- 
ber of  other  microorganisms  have  been  carried  out  by  Dieudonne, 
Janowski,  v.  Esmarch,  and  many  others.  All  these  observers,  while 
differing  somewhat  as  to  the  time  necessary  for  bacterial  destruc- 
tion, agree  in  finding  definite  and  powerful  bactericidal  action  of 
sunlight.  Diffuse  light,  of  course,  is  less  active  than  direct  sunlight. 
According  to  Buchner,  typhoid  bacilli  are  inhibited  by  direct  sun- 
light in  one  and  one-half  hours,  by  diffuse  light  in  five  hours.  A 
remarkable  statement  is  made  by  Arloing,  who  claims  to  have  found 

2  Koch,  Arb.  a.  d.  kais.  Gesundheitsamt,  iii,  1887. 

3  Burkholtz,  Arb.  a.  d.  kais.  Gesundheitsamt,  v,  1889. 

4  Koch,  X  Internat.  Med.  Congress,  Berlin,  1890. 
8  Mignesco,  Arch,  f .  Hyg.,  xxv,  1896. 
'Buchner,  Cent.  f.  Bakt.,  I,  xi,  1892. 
''Arloing,  Compt.  rend,  de  1'acad.  d.  sci.,  c.  1885. 


78  BIOLOGY  AND  TECHNIQUE 

that  anthrax  spores  are  more  quickly  destroyed  by  direct  sunlight 
than  are  the  vegetative  cells.  These  findings  require  further 
confirmation. 

It  has  been  shown  by  various  authors  that  the  influence  of  sun- 
light is  not  to  be  attributed  in  any  way  to  temperature,  nor  always 
to  a  direct  action  of  the  light  upon  the  bacteria,  but  depends  largely 
upon  photochemical  changes  produced  by  the  light  rays  in  the 
media. .  Richardson8  and  Dieudonne9  conclude  that  under  ordinary 
aerobic  conditions  in  fluid  environment  peroxide  of  hydrogen  is 
formed  under  the  influence  of  light.  Novy  and  Freer10  believe  that 
the  bactericidal  effects  in  fluids  noticed  as  a  result  of  exposure  to 
light  are  too  strong  to  be  explained  by  the  formation  of  small 
quantities  of  peroxide  of  hydrogen,  and  attribute  this  action  to 
organic  peroxides  formed  under  the  described  conditions,  such  as 
the  peroxides  of  diacetyl,  benzoylacetyl,  and  others.  These  views 
are  somewhat  strengthened  by  the  fact  that  exclusion  of  oxygen 
from  media  markedly  diminishes  the  bactericidal  power  of  light.11 
That  the  photochemical  changes  alone,  however,  do  not  explain  this 
action  follows  from  the  fact  that  dried  bacteria,  not  surrounded  by 
media,  are  subject  to  a  similar  action.12 

In  analyzing  sunlight  in  regard  to  its  bactericidal  power,  it  has 
been  found  by  various  observers  that  the  most  powerful  action  is 
exerted  by  the  ultraviolet  spectral  rays,  whereas  the  yellow,  red, 
and  ultra-red  rays  are  practically  innocuous.13 

It  is  of  importance  to  note  that  sunlight  has  been  found  also 
to  have  a  strong  attenuating  influence14  upon  some  bacterial  poisons, 
as  shown  by  the  experiments  of  Ferri  and  Celli  upon  tetanus  toxin. 

Electric  light  exerts  a  distinct  bactericidal  action  when  applied 
in  strengths  of  800  to  900  candle  power  for  seven  or  eight  hours.15 

Rontgen  or  X-rays  are  said  by  Zeit,18  Blaise  and  Sambae,17  and 


*  Richardson,  Jour.  Chem.  Soc.,  i,  1893,  Kef.  Deut.  chem.  Gesells.,  xxvi. 

9  Dieudonne,  loc.  cit. 

10  Novy  and  Freer,  3d  Ann.  Meeting  Assn.  Amer.  Bacteriologists,  Chicago,  1901. 
"Eoux,  Ann.  Inst.  Past.,  ix,  1887. 

12  Dieudonne,  loc.  cit. 

13  Ward,  Proc.  Eoyal  Soc.,  52,  1893. 

14  Ferri  and  Celli,  Cent,  f .  Bakt.,  I,  xii,  1892. 

15  Dieudonne,  loc.  cit. 

18  Zeit,  Jour.  Amer.  Med.  Assn.,  xxxvii,  1901. 

17  Blaise  and  Sambae,  Compt.  rend,  de  la  soc.  de  biol.,  1896. 


THE   DESTRUCTION    OF  BACTERIA  79 

others  to  be  without  appreciable  germicidal  power.  Rieder,18  011 
the  other  hand,  has  reported  definite  inhibition  of  bacterial  growth 
after  exposures  of  half  an  hour  to  X-rays. 

Radium  rays  have  a  distinct  inhibitory  and  even  bactericidal 
power  when  applied  at  distances  of  a  few  centimeters  for  several 
hours.19 

Electricity. — If  we  exclude  the  indirect  actions  of  heat  and  elec- 
trolysis, it  can  hardly  be  said  that  the  direct  bactericidal  action  of 
electric  currents  has  been  satisfactorily  demonstrated.  Such  action, 
however,  has  been  claimed  by  d'Arsonville  and  Charrin,20  and  by 
Spilker  and  Gottstein.21 

Heat. — The  most  widely  applicable  and  efficient  physical  agent 
for  sterilization  is  heat. 

The  dependence  of  bacteria  for  growth  and  vitality  upon  the 
maintenance  of  a  proper  temperature  in  their  environment,  and  the 
ranges  of  variation  within  which  bacteria  may  thrive,  have  been 
discussed  in  a  preceding  section,  in  which  a  table  of  so-called 
''thermal  death  points"  has  been  given.  In  the  method  of  express- 
ing these  values  it  was  seen  that  two  elements  entered  into  the 
destruction  of  bacteria  by  heat,  namely,  that  of  the  degree  of  tem- 
perature which  is  applied,  and  that  of  the  time  of  application. 

The  prolonged  application  of  moderately  high  temperatures,  in 
other  words,  may  in  certain  instances,  accomplish  the  same  result 
as  the  brief  use  of  extremely  high  ones.  In  general,  the  death  of 
bacteria  following  prolonged  exposure  to  temperatures  but  slightly 
exceeding  the  optimum  is  due  to  the  inability  of  the  anabolic 
processes  to  keep  pace  with  the  accelerated  katabolic  processes, 
gradual  attenuation  resulting  in  death.  At  somewhat  higher  tem- 
peratures death  results  from  coagulation  of  the  bacterial  protoplasm, 
and  at  still  higher  degrees  of  heat,  applied  in  the  dry  form,  direct 
burning  of  the  bacteria  may  be  the  cause  of  their  destruction. 

Heat  may  be  applied  in  the  form  of  dry  heat  or  as  moist  heat, 
these  methods  being  of  great  practical  value,  but  differently  ap- 
plicable according  to  the  nature  of  the  materials  to  be  sterilized. 
The  two  methods,  moreover,  show  a  marked  difference  in  efficiency, 


18  Eicder,  Munch,  med.  Woeh.,  1898. 
1U  Personal  observations. 

20  D'Arsonville  and  Charrin,  Compt.  rend,  de  la  soc.  de  biol, 

21  Spilker  and  Gottstein,  Cent,  f .  Bakt.,  I,  9,  1891. 


80  BIOLOGY  AND  TECHNIQUE 

temperature  for  temperature.  For  the  recognition  of  this  fact  we 
are  largely  indebted  to  the  early  researches  of  Koch  and  Wolif- 
hiigel,22  and  of  Koch,  Gaffky,  and  Loeffler.23 

These  observers  were  able  to  show  that  the  spores  of  anthrax 
were  destroyed  by  boiling  water  at  100°  C.  in  from  one  to  twelve 
minutes,  whereas  dry  hot  air  was  efficient  only  after  three  hours' 
exposure  to  140°  C.  Extensive  confirmation  of  these  differences  has 
been  brought  by  many  workers.  An  explanation  of  the  phenomena  ob- 
served is  probably  to  be  found  in  the  changes  in  the  coagulability 
of  proteins  brought  about  in  them  by  the  abstraction  of  water. 
Lewith,24  working  with  various  proteins,  found  that  these  substances 
are  coagulated  by  heat  at  lower  temperatures  when  they  contain 
abundant  quantities  of  water,  than  when  water  has  been  abstracted 
from  them.  On  the  basis  of  actual  experiment  with  egg  albumin 
he  obtained  the  following  results,25  which  illustrate  the  point  in 
question  :  , 


Egg  albumin  in  dilute  aqueous  solution,  coagulated  at  56 
"          "          with  25  per  cent  water,  "    74 

«          tt  (t      18    <(       «         «  n  tt    8o 


C. 

74-80°  C. 
tt    8o_90o   C- 


Absolutely  anhydrous  albumin,  according  to  Haas,26  may  be 
heated  to  170°  C.  without  coagulation.  It  is  thus  clear  that  bacteria 
exposed  to  hot  air  may  be  considerably  dehydrated  before  the  tem- 
perature rises  sufficiently  to  cause  death  by  coagulation,  complete 
dehydration  necessitating  their  destruction  possibly  by  actual 
burning. 

Bacteria  exposed  to  moist  air  or  steam,  on  the  other  hand,  may 
absorb  water  and  become  proportionately  more  coagulable. 

The  same  principle,  as  Lewith  points  out,  probably  explains  the 
great  resistance  to  heat  observed  in  the  case  of  the  highly  con- 
centrated protoplasm  of  spores. 

Apart  from  the  actually  greater  efficiency  of  moist  heat  when 
compared  with  dry  heat  of  an  equal  temperature,  an  advantage  of 
great  practical  significance  possessed  by  moist  heat  lies  in  its  greater 

22  Koch  and  Wolff  Mgel,  Mitt.  a.  kais.  Gesundheitsamt,  I,  1882. 

23  Koch,  Gaffky  and  Loeffler,  ibid. 

24  Lewith,  Arch.  f.  exp.  Path.  u.  Pharm.,  xxvi,  1890. 

25  Lewith,  loc.  cit.,  p.  351. 

26  Haas,  Prag.  med.  Woch.,  34-36,  1876. 


THE   DESTRUCTION   OF   BACTERIA  81 

powers  of  penetration.  An  experiment  carried  out  by  Koch  and 
his  associates  illustrates  this  point  clearly.  Small  packages  of  garden 
soil  were  surrounded  by  varying  thicknesses  of  linen  with  thermom- 
eters so  placed  that  the  temperature  under  a  definite  number  of 
layers  could  be  determined.  Exposures  to  hot  air  and  to  steam 
were  then  made  for  comparison,  and  the  results  were  as  tabulated  :27 


Tempera- 
tures. 

Time  of 
Application. 

TEMPERATURES  REACHED  WITHIN 
THICKNESSES  OF  LINEN. 

Twenty 
Thicknesses. 

Forty 
Thicknesses. 

One  Hundred 
Thicknesses. 

Hot  air  
Steam  

130-140°C. 
90-105.3° 

4  hours. 
3  hours. 

86° 
101° 

72° 

101° 

Below  70° 
101.5° 

Incomplete 
steriliza- 
tion. 
Complete 
steriliza- 
tion. 

This  great  penetrating  power  of  steam  is  due  presumably  to  its 
comparatively  low  specific  gravity  which  enables  it  to  displace  air 
from  the  interior  of  porous  materials,  and  also  to  the  fact  that 
as  the  steam  comes  in  contact  with  the  objects  to  be  disinfected  a 
condensation  takes  place  with  the  consequent  liberation  of  heat. 
When  a  vapor  passes  into  the  liquid  state  it  gives  out  a  definite 
amount  of  heat,  which  in  the  case  of  water  vapor,  at  100°  C., 
amounts  to  about  537  calories.  This  brings  about  a  rapid  heating 
of  the  object  in  question.  Following  this  process  the  further  heating 
takes  place  by  conduction,  and  it  is,  of  course,  well  known  that 
steam  is  a  much  better  heat  conductor  than  air.28 

Moist  heat  may  be  applied  as  boiling  water,  in  which,  of  course, 
the  temperature  varies  little  from  100°  C.,  or  as  steam.  Steam  may 
be  used  as  live,  flowing  steam,  without  pressure,  the  temperature 
of  which  is  more  or  less  constant  at  100°  C.,  or  still  higher  efficiency 
may  be  attained  by  the  use  of  steam  under  pressure,  in  which,  of 
course,  temperatures  far  exceeding  100°  C.  may  be  produced,  accord- 
ing to  the  amount  of  pressure  which  is  used. 

The  spores  of  certain  bacteria  of  the  soil  which  can  not  be  killed 
in  live  steam  in  less  than  several  hours  may  be  destroyed  in  a  few 


27  Koch,  Gaffky  und  Loeffler,  loc.  cif.,  p.  339. 

28  Gruber,  Cent,  f .  Bakt.,  iii,  1888. 


82  BIOLOGY  AND  TECHNIQUE 

minutes,  or  even  instantaneously,  in  compressed  steam  at  tempera- 
tures ranging  from  120°  to  140°  C.29 

In  all  methods  of  steam  sterilization,  it  is  of  great  practical 
importance,  as  v.  Esmarch30  lias  pointed  out,  that  the  steam  shall 
be  saturated,  that  is,  shall  contain  as  much  vaporized  water  as  its 
temperature  permits.  Unsaturated,  or  so-called  "super-heated 
steam"  is  formed  when  heat  is  applied  to  steam,  either  by  passage 
through  heated  piping  or  over  heated  metal  plates.  In  such  cases 
the  temperature  of  the  steam  is  raised,  but  no  further  water-vapor 
being  supplied,  the  steam  exerts  less  pressure  and  contains  less  water 
in  proportion  to  its  volume  than  saturated  steam  of  an  equal  tem- 
perature. The  super-heated  steam,  therefore,  is  heated  considerably 
over  its  condensation  temperature  and  becomes  literally  dried.  In 
consequence,  its  action  is  more  comparable  to  hot  air  than  to 
saturated  steam,  and  up  to  a  certain  temperature  its  disinfecting 
power  is  actually  less  than  that  of  live  steam  at  100°  C.  v.  Esmarch, 
who  has  made  a  thorough  study  of  these  conditions,  concludes  that 
up  to  125°  C.,  the  efficiency  of  superheated  steam  is  lower  than  that 
of  live  steam  at  100°  C.  Above  this  temperature,  of  course,  it  is 
again  active  as  in  the  case  of  ordinary  dry  heat. 

PRACTICAL  METHODS  OF  HEAT  STERILIZATION. — Burning. — For  ob- 
jects without  value,  actual  burning  in  a  furnace  is  a  certain  and 
easily  applicable  method  of  sterilization.  Flaming,  by  passage 
through  a  Bunsen  or  an  alcohol  flame,  is  the  method  in  use  for  the 
sterilization  of  platinum  needles,  coverslips,  or  other  small  objects 
which  are  used  in  handling  bacteria  in  the  laboratory. 

Hot  air  sterilization  is  carried  out  in  the  so-called  "hot  air 
chambers,"  simple  devices  of  varied  construction.  The  apparatus 
most  commonly  used  consists  of  a  sheet-iron,  double-walled 
chamber,  the  joints  of  which,  instead  of  being  soldered,  are  closed 
by  rivets.  The  inner  case  of  this  chamber  is  entirely  closed  except 
for  an  opening  in  the  top  through  which  a  thermometer  may  be 
introduced,  while  the  outer  has  a  large  opening  at  the  bottom  and 
two  smaller  ones  at  the  top.  A  gas-burner  is  adjusted  under  this 
so  as  to  play  directly  upon  the  bottom  of  the  inner  case.  A  thermom- 
eter is  fitted  in  the  top  in  such  a  way  that  it  penetrates  into  the 
inner  chamber.  The  air  in  the  chamber  is  heated  directly  by 

28  Christen,  Kef.  Cent,  f .  Bakt.,  V,  xiii,  1893. 
30  v.  Esmarch,  Zeit.  f .  Hyg.,  iv,  1888. 


THE  DESTRUCTION  OF  BACTERIA 


83 


the  flame  and  by  the  hot  air,  which,  rising  from  the  flame,  courses 
upward  within  the  jacket  between  the  two  cases  and  escapes  at  the 
top.  To  insure  absolute  sterilization  of  objects  in  such  a  chamber, 
the  temperature  should  be  kept  between  150°  and  160°  C.  for  at 
least  an  hour.  In  sterilizing  combustible  articles  in  such  a  chamber, 
it  should  be  remembered  that  cotton  is  browned  at  a  temperature 
of  200°  C.  and  over.  .This  method  is  used  in  laboratories  for  the 
sterilization  of  Petri  dishes,  flasks,  test  tubes,  and  pipettes,  and  for 
articles  which  may  be  injured  by  moisture.  Both  heating  and  subse- 
quent cooling  should  be  done  gradually  to  avoid  cracking  of  the 
glassware. 

Moist  Heat. — Instruments,  syringes,  and  other  suitable  objects 
may  be  sterilized  by  boiling  in  water. 
Boiling  for  about  five  minutes  is  amply 
sufficient  to  destroy  the  vegetative  forms 
of  all  bacteria.  For  the  destruction  of 
spores,  boiling  for  one  or  two  hours  is 
usually  sufficient,  though  the  spores  of 
certain  saprophytes  of  the  soil  have  been 
found  occasionally  to  withstand  the  moist 
heat  at  a  temperature  of  100°  C.  for  as 
long  as  sixteen  hours.31  The  addition  of 

1  per  cent  of  sodium  carbonate  to  boiling 
water  hastens  the  destruction  of  spores 
and  prevents  the  rusting  of  metal  objects 
sterilized  in  this  way.     The  addition  of 
carbolic  acid  to  boiling  water  in  from 

2  to  5  per  cent  usually  insures  the  de- 
struction   of    anthrax    spores,    at    least, 
within  ten  to  fifteen  minutes. 

Exposure  to  live  steam  is  prob- 
ably the  most  practical  of  the  methods 
of  heat  sterilization.  It  may  be  carried  out  by  simple  make- 
shifts of  the  kitchen,  such  as  the  use  of  potato-steamers  or  of 
wash-boilers.  For  laboratory  purposes,  the  original  steaming  device 
introduced  by  Koch  has  been  almost  completely  displaced  by  devices 
constructed  on  the  plan  of  the  so-called  "Arnold"  sterilizer  (Fig. 
8).  In  such  an  apparatus,  water  is  poured  into  the  reservoir 


FIG.  8. — ARNOLD  STERILIZER. 


81  Christen,  loe.  cit. 


84  BIOLOGY  AND  TECHNIQUE 

A  and  flows  from  there  into  the  shallow  receptacle  B,  formed  by 
the  double  bottom.  The  flame  underneath  rapidly  vaporizes  the 
thin  layer  of  water  contained  in  B,  and  the  steam  rises  rapidly, 
coursing  through  the  main  chamber  C.  Steam  which  escapes  through 
the  joints  of  the  lid  of  this  chamber  is  condensed  under  the  hood 
and  drops  back  into  the  reservoir.  Exposure  to  steam  in  such  an 
apparatus  for  fifteen  to  thirty  minutes  insures  the  death  of  the 
vegetative  forms  of  bacteria. 

In  the  sterilization  of  media  by  such  a  device,  the  method  of 
fractional  sterilization  at  100°  C.  is  employed.  The  principle  of  this 
method  depends  upon  repeated  exposure  of  the  media  for  fifteen 
minutes  to  one-half  hour  on  three  succeeding  days.  By  the  first 
exposure  all  vegetative  forms  are  destroyed.  The  media  may  then 
be  left  at  room  temperature,  or  at  incubator  temperature  (37.5°  C.) 
until  the  following  day,  when  any  spores  which  may  be  present  will 
have  developed  into  the  vegetative  stage.  These  are  then  killed 
by  the  second  exposure.  A  repetition  of  this  procedure  on  a  third 
day  insures  sterility.  It  must  always  be  remembered,  however,  that 
this  method  is  applicable  only  in  cases  in  which  the  substance  to 
be  sterilized  is  a  favorable  medium  for  bacterial  growth  in  which 
it  is  likely  that  spores  will  develop  into  vegetative  forms. 

Exceptionally  the  method  may  fail  even  in  favorable  media  when 
anaerobic  spore-forming  bacteria  are  present.  Thus,  it  has  been 
observed  that  anaerobic  spores,  failing  to  develop  under  the  aerobic 
conditions  prevailing  during  the  intervals  of  fractional  sterilization, 
have  developed  after  inoculation  of  the  media  with  other  bacteria, 
when  symbiosis  had  made  their  growth  possible.  Tetanus  bacilli 
have,  in  this  way,  occurred  in  cultures  of  diphtheria  bacilli  employed 
for  toxin  production. 

In  noting  the  time  of  an  exposure  in  an  Arnold  sterilizer,  it  is 
important  to  time  the  process  from  the  time  when  the  temperature 
has  reached  100°  C.  and  not  from  the  time  of  lighting  the  flame. 

The  principle  of  fractional  sterilization  at  low  temperatures  is 
applied  also  to  the  sterilization  of  substances  which  can  not  be  sub- 
jected to  temperatures  as  high  as  100°  C.  This  is  especially  the 
case  in  the  sterilization  of  media  containing  albuminous  materials, 
when  coagulation  is  to  be  avoided,  or  when  both  coagulation  of  the 
medium  and  sterilization  are  desired. 

In  such  cases  fractional  sterilization  may  be  practiced  in  simply 
constructed  sterilizers,  such  as  a  Koch  inspissator  or,  in  the  case 


THE  DESTRUCTION  OF  BACTERIA  85 

of  fluids,  such  as  blood  serum,  by  immersion  in  a  water-bath  at  a 
temperature  varying  above  55°  C.,  according  to  circumstances.  Ex- 
posures at  such  low  temperatures  may  be  repeated  on  five  or  six 
consecutive  days,  usually  for  an  hour  each  day. 

The  use  of  steam  under  pressure  is  the  most  powerful  method 
of  heat-disinfection  which  we  possess.  It  is  applicable  to  the 
sterilization  of  fomites,  clothing,  or  any  objects  of  a  size  suitable 
to  be  contained  in  the  apparatus  at  hand,  and  which  are  not  injured 
by  moisture.  In  laboratories  this  method  is  employed  for  the 
sterilization  of  infected  apparatus,  such  as  flasks,  test  tubes,  Petri 
plates,  etc.,  containing  cultures.  The  device  most  commonly  used 
in  laboratories  is  the  so-called  autoclave,  of  which  a  variety  of 
models  may  be  obtained,  both  stationary  and  portable.  The  prin- 
ciple governing  the  construction  of  all  of  these  is  the  same.  The 
apparatus  usually  consists  of  a  gun-metal  cylinder  supplied  with  a 
lid,  which  can  be  tightly  closed  by  screws  or  nuts,  and  supplied 
with  a  thermometer,  a  safety-valve,  and  a  steam  pressure  gauge. 
In  the  simpler  autoclaves,  water  may  be  directly  filled  into  the  lower 
part  of  the  cylinder,  and  the  objects  to  be  sterilized  supported  upon 
a  perforated  diaphragm.  In  this  case  the  heat  is  directly  applied 
by  means  of  a  gas  flame.  In  the  more  elaborate  stationary  devices, 
steam  may  be  let  in  by  piping  it  from  the  regular  supply  used  for 
heating  purposes.  Exposure  to  steam  under  fifteen  pounds  pressure 
(fifteen  in  addition  to  the  usual  atmospheric  pressure  of  fifteen 
pounds  to  the  square  inch)  for  fifteen  to  twenty  minutes,  is  sufficient 
to  kill  all  forms  of  bacterial  life,  including  spores. 

In  applying  autoclave  sterilization  practically,  attention  must  be 
paid  to  certain  technical  details,  neglect  of  which  would  result  in 
failure  of  sterilization.  It  is  necessary  always  to  permit  all  air  to 
escape  from  the  autoclave  before  closing  the  vent.  If  this  is  not 
done,  a  poorly  conducting  air-jacket  may  be  left  about  the  objects 
to  be  sterilized,  and  these  may  not  be  heated  to  the  temperature 
indicated  by  the  pressure.  It  is  also  necessary  to  allow  the  reduction 
of  pressure,  after  sterilization,  to  take  place  slowly.  Any  sudden 
relief  of  pressure,  such  as  would  be  produced  by  opening  the  air- 
vent  while  the  pressure  gauge  is  still  above  zero,  will  usually  result 
in  a  sudden  ebullition  of  fluid  and  a  removal  of  stoppers  from  flasks. 

The  temperature  attained  by  the  application  of  various  degrees 
of  pressure  is  expressed  in  the  following  table : 


86  BIOLOGY  AND  TECHNIQUE 

Lbs.  Pressure  Temperature   j   Lbs.  Pressure  Temperature 

1   102.3°C.   I          13 119.10. 

.    104.2  14   .  .    120.2 


3 105.7 

4 107.3 

5  108.8° 

6  110.3 

7  111.7 

8  113 

9  114.3( 

10 115.6 

11  116.8 

12  .  .   118 


15   121.3 

16   122.4 

17   123.3° 

18    124.3 

20   126.2 

22   128.1 

24 129.3 

26   131.5 

28   133.1 

30   .  .  134.6 


CHEMICAL   AGENTS    INJURIOUS   TO    BACTERIA 

Since  the  time  of  Koch's32  fundamental  researches  upon  chemical 
disinfectants,  the  known  number  of  these  substances  has  been 
enormously  increased,  and  now  embraces  chemical  agents  of  the 
most  varied  constitution.  It  is  thus  manifestly  impossible  to  refer 
the  injurious  influence  which  these  substances  exert  upon  bacteria 
to  any  uniform  law  of  action.  The  efficiency  of  a  disinfecting  agent, 
furthermore,  is  not  alone  dependent  upon  the  nature  and  concen- 
trations of  the  substance  itself,  but  depends  complexly  upon  the 
nature  of  the  solvent  in  which  it  is  employed,  the  temperature  pre- 
vailing during  its  application,  the  numbers  and  biological  character- 
istics of  the  bacteria  in  question,  and  the  time  of  exposure.  All 
these  factors,  therefore,  must  be  considered  in  testing  the  efficiency 
of  any  given  disinfectant.  While  it  is  true,  furthermore,  that  all 
substances  which  in  a  given  concentration  exert  bactericidal  or 
disinfecting  action  upon  a  microorganism,  will  in  greater  dilution 
act  antiseptically  or  inhibitively,  no  definite  rules  of  proportion  exist 
between  the  two  values,  which  in  each  case  must  be  determined 
by  experiment. 

Disinfectants  Used  in  Solution. — The  actual  processes  which  take 
place  in  the  injury  of  bacteria  by  disinfectants  are  to  a  large  extent 
unknown.  In  the  ease  of  strong  acids,  or  strongly  oxidizing  sub- 
Stances,  there  may  be  destruction  of  the  bacterial  body  as  a  whole 
by  rapid  oxidation.  Other  substances  may  act  by  coagulation  of 
the  bacterial  protoplasm;  others  again  by  diffusion  through  the 
cell  membrane  are  able  to  enter  into  chemical  combination  with  the 


82  Koch,  Arb.  a.  d.  kais.  Gesundheitsamt,  i,  1881. 


THE  DESTRUCTION  OF  BACTERIA  87 

protoplasm  and  exert  a  toxic  action.  Again,  in  other  cases,  a  dif- 
ference in  tonicity  between  cell  protoplasm  and  disinfectant  may 
tend  to  withdrawal  of  water  from  the  bacterial  cell  and  consequent 
injury  of  the  microorganism. 

Among  the  inorganic  disinfectants  the  most  important  are  the 
metallic  salts,  acids,  and  bases,  the  halogens  and  their  derivatives, 
and  certain  oxidizing  agents  like  peroxide  of  hydrogen  and  perman- 
ganate of  potassium. 

It  has  been  shown  by  Scheuerlen  and  Spiro,33  Kronig  and  Paul,3* 
and  others,  that  in  the  case  of  the  salts,  acids,  and  bases,  there  is 
a  distinct  and  demonstrable  relationship  between  the  disinfecting 
power  of  these  substances  and  their  dissociation  in  solution. 

According  to  the  theory  of  electrolytic  dissociation,  when  bodies 
of  this  class  go  into  solution  they  are  broken  up  or  dissociated  into 
an  electro-positive  and  an  electro-negative  ion.  Thus,  metallic  salts 
are  broken  up  into  the  kation,  or  positive  metal,  and  into  the  anion, 
or  negative  acid  radicle  (AgN03  =  Ag,  +  ion  and  N03,  --  ion). 
In  the  case  of  the  acids,  ionization  takes  place  into  the  hydrogen 
ions  and  the  acid  radicles,  while  in  the  case  of  the  bases  the  dissocia- 
tion occurs  into  the  metal,  on  the  one  hand,  and  the  OH  group  on 
the  other.  The  degree  of  dissociation  taking  place  depends  upon 
the  nature  of  the  substance  in  solution,  its  concentration,  and  the 
nature  of  the  solvent.  Thus,  in  any  such  solution  there  appear  three 
substances,  the  undissociated  compound  as  such,  its  electro-negative 
ion,  and  its  electro-positive  ion,  their  relative  concentrations  depend- 
ing upon  an  interrelationship  calculable  by  definite  laws.  It  goes 
without  saying,  therefore,  that  any  chemical  or  physical  reaction, 
taken  part  in  by  such  a  solution,  may  be  participated  in,  not  only 
by  the  dissolved  iindissociated  residue  as  a  whole,  but  by  its  separate 
ions  individually  as  well.  In  the  case  of  many  disinfectants,  the 
writers  referred  to  above  have  been  able  to  demonstrate  a  relation- 
ship between  the  degree  of  dissociation  and  the  bactericidal  powers. 
According  to  Kronig  and  Paul,  double  metallic  salts,  in  which  the 
metal  is  a  constituent  of  a  complex  ion  and  in  which  the  concentration 
of  the  dissociated  metal-ions  is  consequently  low,  have  very  little 
disinfecting  power.  Thus  potassium-silver-cyanide,  which  is  a  com- 
paratively weak  disinfectant,  dissociates  into  the  kation  K  and  the 


33  Scheuerlen  und  Spiro,  Munch,  med.  Woch.,  44,  1897. 
84  Krdnig  un<l  Paul,  Zeit.  f .  Hyg.,  xxv,  1897. 


88  BIOLOGY  AND  TECHNIQUE 

complex  anion  Ag  (CN)2,  this  latter  further  dissociating  to  a  very 
slight  degree  only.  The  same  writers  conclude  that  the  bactericidal 
action  of  mercuric  chloride  and  of  halogen  combinations  with  metals 
is  directly  proportionate  to  the  degree  of  dissociation.  This  con- 
sideration, moreover,  explains  why  aqueous  solutions  of  such  sub- 
stances are  more  active  than  are  solutions  in  the  alcohols  or  in 
ether,  since  it  is  well  known  that  metallic  salts  are  ionized  in  these 
substances  to  a  much  slighter  degree  than  they  are  in  water.35 

On  the  other  hand,  the  addition  of  moderate  quantities  of  ethyl 
and  methyl  alcohol  or  acetones  to  aqueous  solutions  of  silver  nitrate 
or  mercuric  chloride,  definitely  increases  the  disinfecting  action  of 
such  solutions.  In  the  case  of  mercuric  chloride,  Kronig  and  Paul 
obtained  the  most  powerful  effects  in  solutions  to  which  alcohol  had 
been  added  in  a  concentration  of  25  per  cent.  For  this  empirical 
fact  a  satisfactory  explanation  has  not  yet  been  found.  Kronig 
and  Paul  suggest  that  low  percentages  of  alcohol  may  facilitate 
the  penetration  of  the  disinfectant  through  the  cell  membrane  and 
thus  increase  its  efficiency,  while  high  percentages  of  alcohol  have 
the  opposite  effect,  by  decreasing  the  degree  of  dissociation.  In 
this  connection  it  has  been  suggested,  however,  that  absolute  and 
strong  alcohols  possibly  act  as  desiccating  agents,  thus  actually 
rendering  the  bacteria  dry  and  less  susceptible  to  deleterious 
chemical  influences. 

In  the  case  of  acids  and  bases  the  same  authors  have  determined 
that  the  powers  of  disinfection  of  these  substances  are  again  directly 
proportionate  to  the  degree  of  their  dissociation:  that  is,  to  the 
concentration  of  the  hydrogen  or  hydroxyl  ions,  respectively.  The 
hydrogen  ions  are  more  powerfully  active  than  the  hydroxyl  ions 
in  equal  concentration;  acids,  therefore,  are  more  efficient  disin- 
fectants than  bases. 

A  fact  which  appears  to  strengthen  the  opinion  as  to  the  relation- 
ship between  bactericidal  powers  and  dissociation,  is  that  brought 
forward  by  Scheuerlen  and  Spiro,  that  the  addition  of  NaCl  to 
bichloride  of  mercury  solutions  reduces  the  disinfecting  power  of 


*5  Water  is  the  strongest  'dissociant  known.  Methyl  alcohol  has  about  one-half 
to  two-thirds  the  dissociating  power  of  water  (ZelinsTcy,  Zeit.  f.  physiol.  Chemie, 
xx,  1896).  Ethyl  alcohol  allows  dissociation  much  less  than  methyl  alcohol; 
ammonia  allows  dissociation  to  about  one-third  to  one-fourth  the  extent  of  water. 
See  Jones,  "Elements  of  Physical  Chemistry,"  p.  371.  Macmillan,  New  York, 
1902. 


THE   DESTRUCTION  OF  BACTERIA  89 

such  solutions,  inasmuch  as  it  diminishes  the  concentration  of  free 
ions.  In  practice,  however,  NaCl  or  NH4C1  is  added  to  bichloride 
of  mercury  solutions,  since  these  substances  aid  in  holding  in  solu- 
tion mercury  compounds  formed  in  the  presence  of  alkaline  al- 
buminous material,  blood  serum,  pus,  etc. 

The  principles  underlying  disinfection  have  been  still  further 
elucidated  by  Chick,36  who  showed  that  the  rate  at  which  bacteria 
were  killed  followed  the  definite  mathematical  expression  for  velocity 
of  simple  chemical  reactions  of  the  monomolecular  type.  It  was 
found  necessary  to  express  the  concentration  of  ionized  antiseptics 
in  terms  of  concentration  of  the  active  ion  (Hg)  instead  of  total 
molecular  concentration,  thus  adducing  further  verification  of  the 
work  of  Scheuerlen  and  Spiro,  and  Kronig  and  Paul.  There  was 
also  found  to  be  an  unusually  high  temperature  coefficient  for  dis- 
infectant action,  in  the  case  of  phenol  the  reaction  velocity  increas- 
ing eight  times  for  a  rise  of  10°  C.,  while  with  metallic  salts,  the 
increase  was  about  three-fold.  The  advantage  to  be  gained  by  the 
use  of  warm  solutions  is,  therefore,  evident. 

Halogens.* — In  regard  to  the  halogens,  Kronig  and  Paul  have 
shown  that  the  germicidal  power  of  this  class  of  elements  is  inversely 
proportionate  to  their  atomic  weights.  Thus,  chlorine  with  the 
lowest  atomic  weight  is  the  strongest  disinfectant  of  the  group. 
Next,  and  almost  equal  to  this,  is  bromine.  Iodine  with  a  much 
heavier  atomic  weight  than  either  of  the  former  is  distinctly  less 
bactericidal. 

CHLORIDE  OF  LIME. — Of  the  halogen  compounds  used  in  practice, 
the  most  important  is  chloride  of  lime  or  bleaching  powder.  As  to 
the  composition  of  this  substance,  there  is  some  difference  of  opinion. 
It  was  formerly  believed  to  be  a  mixture  of  calcium  hypochlorite, 
Ca(C102),  and  of  calcium  chloride,  CaCl2.  The  fact  that  the  sub- 
stance is  not  deliquescent,  however,  speaks  against  the  presence  of 
calcium  chloride  as  such,  and  it  is  probable  that  it  consists  of  a 
single  compound  with  the  formula  CaOCl2.  The  action  of  acids  or 
even  of  atmospheric  C02  upon  this  substance  results  In  the  liberation 
of  chlorine.  For  instance : 

Ca(Cl20)  +  2HC1  =  CaCl2  +  2HC10. 
_  2HC10       +  2HC1  =  2H,0  +  2C12. 

"Chick,  Jour,  of  Hyg.,  8,  1908,  92,  and  10,  1910,  238. 

*  For  consideration  of  the  uses  of  chlorin  and  bleaching  powder  for  the  treat- 
ment of  drinking  water,  see  Chapter  on  "Water." 


90  BIOLOGY  AND  TECHNIQUE 

Hypochlorous  acid  may  also  decompose  with  the  liberation  of 
oxygen  as  shown  in  the  following  equation: 

2HC10  ==  2HC1  +  02 

It  is  conceivable  that  some  of  the  disinfecting  value  of  chloride  of 
lime  and  hypochlorites  in  general  is  really  due  to  the  vigorous 
oxidizing  action  resulting  from  this  decomposition.  On  the  other 
hand,  there  is  much  evidence  to  show  that  chlorine  may  attack  the 
protein  molecule  directly  by  replacing  "H"  in  the  amino  groups, 
thus: 

_R__CO  —  NH  —  R  —    +  C1  =  R—  CO  —  NCI—  R-      +H 

The  chloramines  thus  formed  seem  to  be  toxic  and  result  in  the 
death  of  the  bacteria.  Bleaching  powder  is  readily  soluble  in  about 
twenty  parts  of  water.  Its  bactericidal  action  depends  on  the 
hypochlorous  acid  formed.  After  water  precipitation,  an  efficient 
dosage  is  10  pounds  to  the  million  gallons.  The  high  germicidal 
action  of  chloride  of  lime,  together  with  its  relatively  low  cost, 
suggest  its  use  as  a  wound  dressing.  Solutions  of  calcium  or  sodium 
hypochlorite  were  found  to  be  too  irritating  to  be  practicable,  owing 
to  the  alkalinity  of  any  available  preparations.  Recently,  however, 
it  has  been  possible  to  prepare  neutral,  and  comparatively  non- 
irritating  solutions  of  sodium  hypochlorite  by  several  different 
methods. 

Dakins  Solution. — The  following  detailed  descriptions  of  the  prepara- 
tion of  Dakin's  solution  and  its  titration  are  taken  from  the  Medical  War 
Manual,  Number  6,  Laboratory  Methods  of  the  United  States  Army,  published 
from  the  Surgeon  General's  Office,  and  are  given  directly  as  printed  in  this 
Manual,  because  the  method  was  standardized  for  Army  use  in  this  way 
during  the  war.  The  description  which  follows  is  taken  verbatim  from 
this  Manual. 

Preparation  from  Bleaching  Powder. — Dakin's  Original  Method. — A 
strong  solution  of  hypochlorite  is  prepared  by  decomposing  150  grams 
bleaching  powder  (about  25  to  35  per  cent  available  chlorine)  with  105 
grams  dry  sodium  carbonate  (122  grams  monoliydrate  (Na.,CO,H,0)  or  284 
grams  washing  soda  (Na2C0810H20).  The  mixture  is  very  thoroughly 
shaken,  both  to  make  good  contact  and  to  render  the  precipitated  calcium 
carbonate  granular  and  promote  its  settling.  It  is  then  allowed  to  stand 
quietly  and  after  half  an  hour  the  clear  liquid  is  siphoned  off  from  the 
precipitate  and  filtered  through  paper  or  a  cotton  plug. 


THE  DESTRUCTION  OF  BACTERIA  91 

N 
A  10  c.c.  portion  is  rapidly  titrated  with — boric  acid  solution  (31  grams 

2i 

per  liter),  using  powdered  phenolplithalein  as  indicator  (the  usual  alcoholic 
solution  of  phenolphthalein  will  not  serve)  in  order  to  determine  the  amount 
of  boric  acid  to  be  added  to  the  rest  of  the  filtrate.  The  end-point  is  the 

N 
disappearance  of  the  pink  color.     Each  cubic  centimeter  of  -  -   boric  acid 

2i 

required  for  the  10  c.c.  sample  calls  for  the  addition  of  3  grains  boric  acid 
per  liter  of  filtrate.  An  excess  of  boric  acid  should  be  avoided,  as  it  favors 
the  liberation  of  hypochlorous  acid  and  renders  the  solution  less  stable.  It 
is  best  to  add  slightly  less  than  the  calculated  amount.  The  concentrated 
solution  thus  prepared  contains  about  4  per  cent  of  sodium  hypochlorite, 
and  before  use  should  be  diluted  with  about  7  parts  of  water  and  titrated 

N 
with  —  thiosulphate  to  determine  its  precise  hypochlorite  concentration.     It 

10 

is  then  accurately  diluted  to  the  required  strength  (usually  0.5  to  0.45  per 
cent). 

Preparation  from  Chlorine  and  Sodium  Carbonate. — Chlorine  may  be  ob- 
tained as  the  compressed  gas  in  steel  cylinders  and  is  easily  measured  by 
a  chlorine  meter  manufactured  for  the  purpose.  This  is  a  stable,  economical 
and  convenient  source  of  chlorine.  A  solution  is  prepared  containing  15 
grams  of  dry  sodium  .carbonate  per  liter  (=17.6  grams  monohydrate  or 
40  grams  of  washing  soda).  A  measured  quantity,  4.8  grams  (or  about 
1600  c.c.)  of  chlorine  gas  is  allowed  to  run  into  the  solution  for  each  liter. 
Ten  c.c.  of  the  solution  is  then  titrated.  If  the  solution  is  too  weak  more 
chlorine  is  introduced.  If  the  solution  is  too  strong  it  should  be  diluted 
to  a  concentration  of  0.5  per  cent  NaOCl  with  1.5  per  cent  sodium  carbonate 
solution,  which  at  the  same  time  serves  to  correct  the  unduly  diminished 
alkalinity  caused  by  the  excess  of  chlorin  introduced  into  the  solution.  If 
the  final  solution  fails  to  give  a  momentary  flash  of  color  with  alcoholic 
solution  of  phenolphthalein,  it  should  be  rejected.  If  the  solution  shows 
color  with  powdered  phenolphthalein,  it  must  be  titrated  with  boric  acid  as 
described  above,  for  preparation  from  bleaching  powder,  until  this  defect 
is  corrected,  or  it  must  be  discarded.  The  solution  should  be  titrated  for 
hypochlorite  concentration  every  twenty-four  to  forty-eight  hours  and  dis- 
carded when  it  falls  below  the  desired  lower  limit  (usually  0.45  per  cent). 

If  a  chlorin  meter  is  not  available,  chlorin  gas  may  be  run  into  the 
1.5  per  cent  carbonate  solution  through  any  improvised  diffuser  until  the 
hypochlorite  concentration  has  reached  0.5  per  cent.  The  amount  of  chlorin 
required  to  give  a  hypochlorite  concentration  of  0.5  per  cent  is*  approximately 
twice  the  amount  required  to  cause  decolorization  of  powdered  phenol- 
phthalein. It  is  therefore  convenient  to  add  powdered  phenolphthalein  and 
note  the  amount  of  chlorin  required  to  cause  its  decolorization.  When 


92  BIOLOGY  AND  TECHNIQUE 

almost  twice  that  amount  of  chlorin  has  been  introduced,  frequent  titrations 
of  the  hypochlorite  content  must  be  commenced  to  determine  the  proper 
point  at  which  to  stop  the  introduction  of  the  chlorin. 

Titration  of  Dakin's  Solution. — To  10  c.c.  of  the  Dakin  solution,  add 
approximately  5  c.c.  of  a  10  per  cent  solution  of  potassium  iodid  and  3  c.c. 
of  glacial  acetic  acid.  lodin  is  liberated  and  dissolves  in  the  excess  of  iodid 

N 
present.     Dilute  with  water  to  about  50  c.c.     A  standard   --  thiosulphate 

solution  is  then  added  from  a  burette  until  the  solution  is  just  rendered 
colorless.  The  number  of  cubic  centimeters  required  to  effect  this  result 
multiplied  by  the  factor  0.0372  gives  the  percentage  of  sodium  hypochlorite 
present  in  the  Dakin  solution. 

N 
Preparation    of   Standard  Thiosulphate    Solution. — Dissolve    exactly 

24.82  grams  of  pure  crystalline  sodium  thiosulphate  in  water  and  make 
up  to  exactly  1  liter.  One  c.c.  of  this  standard  solution  is  equivalent  to 
0.00372  gram  of  sodium  hypochlorite. 

Titration  of  Bleaching  Powder. — Bleaching  powders  vary  considerably  in 
their  "available  chlorin"  content,  so  that  it  is  desirable  to  determine  the 
available  chlorine  in  each  large  batch.  Bleaching  powders  with  less  than 
20  per  cent  available  chlorin  should  be  rejected.  Exceptional  samples  may 
contain  as  high  as  35  per  cent  available  chlorine. 

The  available  chlorin  content  may  be  determined  as  follows:  exactly 
10  grams  of  bleaching  powder  made  up  of  small  samples  from  different 
parts  of  the  jar,  in  order  to  obtain  a  representative  sample,  are  well  shaken 
with  a  liter  of  water.  After  standing  about  six  hours  the  solution  is  filtered 
and  a  10  c.c.  sample  of  the  filtrate  is  titrated  in  exactly  the  same  manner 
as  in  the  titration  of  Dakin's  solution.  In  this  case  the  number  of  cubic 
centimeters  of  decinormal  thiosulphate  required  to  decolorize,  multiplied  by 
the  factor  3.55,  gives  the  percentage  of  active  chlorin  in  the  bleaching 
powder. 

The  chlorin  antiseptics  in  general,  and  particularly  the  hypo- 
chlorite type,  have  the  disadvantage  of  exerting  their  disinfectant 
action  over  an  exceedingly  short  space  of  time.  The  reaction  be- 
tween the  hypochlorite  solution  and  the  proteins  of  the  bacterial  body 
or  of  the  serum  and  pus  in  the  wound,  is  almost  instantaneous,  and 
having  taken  place,  no  further  toxic  action  is  shown.  It  is,  there- 
fore, necessary  in  treating  wounds  with  these  solutions,  to  repeat 
the  application  at  frequent  intervals,  or  else  to  apply  through  some 
sort  of  a  continuous  feed  apparatus  so  that  a  fresh  supply  of  the 
antiseptic  is  brought  in  contact  with  the  wound  at  short  intervals. 

In  order  to  overcome  this  disadvantage  to  some  extent,  DaHn 


THE   DESTRUCTION  OF  BACTERIA  93 

prepared  a  number  of  different  organic  chlorin  compounds  which 
were  soluble  in  oil,  and  which  yielded  up  the  chlorin  rather  slowly 
to  the  wound  secretions,  so  that  the  action  continued  over  a  com- 
paratively long  time.  Chloramine  T  (1)  and  Dichloramine  T  are 
the  two  most  practicable  compounds ;  these  substances  have  the  fol- 
lowing formulae : 

CH3 


SO2NaNCl  SO2NC12 

Chloramine-T  Dichloramine-T 

They  contain  chlorin  replacing  hydrogen  in  an  amino  group,  and 
this  chlorin  is  liberated  slowly,  in  contact  with  protein  material. 
They  are  used  in  solution  in  oil,  either  chlorinated  paraffin  oil  or  oil 
of  eucalyptol,  and  are  applied  as  a  spray  or  on  gauze. 

Eusol. — The  simple  neutralization  of  calcium  hypochlorite  with 
boric  acid  renders  it  comparatively  non-irritating,  and  under  the 
name  of  eusol  (2)  this  solution  has  been  widely  used. 

For  original  articles  on  the  use  of  Dakin 's  Solution,  see  below: 37 

TERCHLORIDE  OF  IODIN  (IC13)  is  an  extremely  strong  disinfectant, 
being  efficient  for  vegetative  forms  in  solutions  of  0.1  per  cent  in 
one  minute  and  a  1  per  cent  solution  destroying  spores  within  a  few 
minutes.38 

Painting  with  TINCTURE  OF  IODIN  (10  per  cent)  is  a  simple  and 
reliable  method  of  sterilizing  the  skin.  It  is  now  used  in  many 
clinics  in  sterilizing  the  field  of  operation. 

IODOFORM  (CHI3)39  is  weakly  antiseptic  in  itself,  but  when  in- 
troduced into  wounds  where  active  reducing  processes  are  taking 
place — often  as  the  result  of  bacterial  growth — iodine  is  liberated 
from  it  and  active  bactericidal  action  results. 

PEROXIDE  OF  HYDROGEN  is  formed  by  the  action  of  dilute  sulphuric 

87  Dakin,  Brit.  Med.  Jour.,  August  28th,  1915.  Carrel,  Dakin,  Daufresne, 
Dehelly  and  Dumas,  Presse  Medicale,  October  llth,  1915.  Dakin  and  Dun- 
ham, " Handbook  of  Antiseptics,"  New  York,  1917.  Lorrain  Smith,  Dren- 
nan,  Eettie  and  Campbell,  British  Med.  Jour.,  July  24th,  1915.  See  also  Carrel 
and  Dehelly,  "Infected  Wounds,"  Hoeber,  N.  Y.  1919. 

3sBehring,  Zeit.  f.  Hyg.,  ix,  1891. 

39  v.  Behring,  <  <  Bekaempf ung  d.  Inf ektions-Krankh., "  Leipzig,  1894. 


94  BIOLOGY  AND  TECHNIQUE 

acid  upon  peroxide  of  barium.  It  readily  gives  up  oxygen  and  acts 
upon  bacteria  probably  by  virtue  of  the  liberation  of  nascent  oxygen. 
In  the  presence  of  organic  matter,  such  as  blood,  pus,  etc.,  associated 
with  bacteria,  H202  is  quickly  reduced  and  weakened.  It  is  impor- 
tant that  the  H202  come  in  immediate  contact  with  the  bacteria. 
In  practice,  therefore,  blood  and  pus  should  be  removed  from  wounds 
when  applying  the  H202  or  a  large  excess  of  H202  should  be  used. 

PERMANGANATE  OP  POTASSIUM,  acting  probably  in  the  same  way, 
is  a  powerful  germicide.  It  also  is  readily  reduced  by  many  organic 
substances  often  associated  with  bacteria,  being  rendered  weaker 
thereby. 

Among  organic  disinfectants  those  of  most  practical  importance 
are  the  alcohols,  formaldehyds,  iodoform,  members  of  the  phenol 
group  and  its  derivatives,  carbolic  acid,  cresol,  lysol,  creolin,  sali- 
cylic acid,  certain  ethereal  oils,  and,  more  recently  introduced, 
organic  silver  salts  such  as  protargol,  argyrol,  argonin,  and  others. 

THE  ALCOHOLS  are  but  indifferent  disinfectants.  Koch40  in  1881 
found  that  anthrax  spores  remained  alive  for  as  long  as  four  months 
when  immersed  in  absolute  and  in  50  per  cent  ethyl  alcohol.  On  the 
other  hand,  while  absolute  alcohol  possesses  practically  no  germicidal 
powers,  possibly  because  of  the  formation  of  a  protecting  envelope 
by  the  coagulation  of  the  bacterial  ectoplasm,  or,  as  suggested  above, 
by  desiccation  due  to  the  abstraction  of  water,  dilute  alcohol  in  a 
concentration  of  about  50  per  cent  is  distinctly  germicidal,  destroy- 
ing the  vegetative  forms  of  bacteria  in  from  ten  to  fifteen  minutes 
or  less.41  Attention  has  already  been  called  to  the  fact  that  moderate 
additions  of  alcohol  to  aqueous  solutions  of  mercuric  chloride  en- 
hance the  germicidal  power  of  this  disinfectant.  Additions  of  ethyl 
and  methyl  alcohol  to  carbolic  acid  or  formaldehyde  solutions,  on 
the  other  hand,  progressively  decrease  the  bactericidal  activities  of 
these  substances.42 

The  value  of  boiling  alcohol  for  the  destruction  of  spores— 
especially  in  the  sterilization  of  catgut — has  been  investigated  by 
Saul,43  who  found  that  boiling  in  absolute  ethyl,  methyl,  or  propyl 
alcohol  is  practically  without  effect,  while  spores  are  destroyed 


40  Koch,  Arb.  a.  d.  kais.  Gesundheitsamt,  i,  1881. 

41  Epstein,  Zeit.  f .  Hyg.,  xxiv,  1897. 
*-  Krdnig  und  Paul,  loc.  cit. 

43  Saul,  Archiv  f .  klin.  Chir.,  56,  1898, 


THE   DESTRUCTION   OF   BACTERIA  95 

readily  in  boiling  dilute  alcohol,  the  most  effectual  being  propyl 
alcohol  of  a  concentration  of  from  10-40  per  cent. 

CARBOLIC  ACID  (C6H5OH),  at  room  temperature,  consists  of  color- 
less crystals  which  become  completely  liquefied  by  the  addition  of 
10  per  cent  of  water.  In  contradistinction  to  most  inorganic  dis- 
infectants, the  action  of  carbolic  acid  and  other  members  of  the 
phenol  group  is  not  in  any  way  dependent  upon  dissociation.44 
According  to  Beckmann45  and  others,  carbolic  acid  acts  as  a  mole- 
cule and  not  by  individual  ions.  The  proof  of  this  is  brought  out 
by  the  fact  that  the  addition  of  NaCl  to  carbolic  acid  solutions, 
an  addition  which  would  tend  to  decrease  the  concentration  of  free 
ions,  markedly  increases  the  bactericidal  powers  of  such  solutions. 
On  the  other  hand,  as  stated  above,  additions  of  alcohol  progres- 
sively diminish  the  efficiency  of  the  phenols. 

Other  members  of  this  group  of  disinfectants  are  ORTHO-,  META-, 
and  PARACRESOL  (C6H4CH3OH),  isomeric  compounds  differing  only 
in  the  position  of  the  OH  radicle.  Tricresol  is  a  mixture  of  these 
three.  The  cresols  are  relatively  more  powerfully  germicidal  than 
carbolic  acid,  but  are  less  soluble  in  water.  LYSOL  is  a  substance 
obtained  by  the  solution  of  coal-tar  cresol  in  neutral  potassium-soap. 
Dissolved  in  water  it  forms  an  opalescent  easily  flowing  liquid. 
According  to  Gruber,46  its  germicidal  action  is  slightly  greater  than 
that  of  carbolic  acid.  CREOLIN,  another  combination  of  the  cresols 
with  potassic  soap,  forms  with  water  a  turbid  emulsion,  v.  Behring47 
expressed  the  relative  germicidal  powers  of  carbolic  acid,  cresol, 
and  creolin  for  vegetative  forms  by  the  numbers  1  :  4  :  10,  in  the 
order  named. 

FORMALDEHYD  (H-COH),  or  methyl  aldehyde,  is  a  gas  which  is 
easily  produced  by  the  incomplete  combustion  of  methyl  alcohol. 
The  methods  of  actually  generating  it  for  purposes  of  fumigation 
will  be  discussed  in  a  subsequent  paragraph.  In  aqueous  solution 
this  substance  forms  a  colorless  liquid  with  a  characteristic  acrid 
odor,  and  in  this  form  is  Jargely  used  as  a  preservative  for  animal 
tissues  and  as  a  germicide.  It  is  marketed  as  "formalin,"  which 
is  an  aqueous  solution  containing  from  35  to  40  per  cent  of  the 


44  Scheuerlen  und  Spiro,  Munch,  med.  Woch.,  44,  1897. 

45  BecTcmann,  Cent,  f .  Bakt.,  I,  xx,  1896. 
48  Gruber,  Cent.  f.  Bakt,  I.,  xi,  1892. 
"v.  Behring,  loc.  cit.,  p.  111. 


96  BIOLOGY  AND  TECHNIQUE 

gas  and  which  exerts  distinctly  bactericidal  action  on  vegetative 
forms  in  further  dilutions  of  of  from  1  to  10  to  1  to  20  (formaldehyd 
gas  1  :  400  to  1  :  800).  Anthrax  spores  are  killed  in  35  per  cent 
formaldehyde  in  ten  to  thirty  minutes.48  Unlike  the  phenols,  the 
addition  of  salt  to  formaldehyd  solutions  does  not  increase  its 
efficiency,  but  similar  to  them,  additions  of  ethyl  and  methyl  alcohol 
markedly  reduce  its  germicidal  powers. 

THE  ESSENTIAL  OILS  which  are  most  commonly  used  in  practice — 
largely  as  intestinal  antiseptics — are  those  of  cinnamon,  thyme, 
eucalyptus,  and  peppermint.  Omeltschenko49  believes  that  the  em- 
ployment of  these  oils  in  emulsions  is  illogical,  inasmuch  as  their 
bactericidal  powers  depend  upon  their  vaporization.  He  classifies  the 
oils  in  decreasing  order  of  their  efficiency  as  follows:  Oil  of  cinna- 
mon, prunol,  oil  of  thyme,  oil  of  peppermint,  oil  of  camphor,  and 
eucalyptol. 

The  Flavine  Dyes. — A  number  of  dye  stuffs  have  been  shown  to 
exert  disinfectant  action  when  applied  to  bacteria.  The  members 
of  the  acridine  group  are  particularly  active  in  this  respect,  and 
have  been  used  to  some  extent  practically.  Benda50  first  investigated 
one  of  these  dyes,  now  known  under  the  name  of  flavine  or  acri- 
flavine,  in  connection  with  trypanosome  infections,  and  recently 
Browning51  applied  this  substance  to  the  antiseptic  treatment  of 
war  injuries. 


NH2 


CH3  Cl 

Acriflavine. 

He  believed  that  this  dye,  while  being  highly  toxic  to  bacteria,  was 
relatively  harmless  and  non-irritating  in  its  effect  on  the  tissue  cells. 


48  Kronig  und  Paul,  loe.  cit. 

49  Omeltschenko,  Cent.  f.  Bakt.,  I,  ix,  1891. 

50  Benda,  Ber.  Deutseh.  Chem.  Gesell,  45,  1787,  1912. 

51  Browning,  Gulbsansen,  Kennaway  and  Thornton,  Brit.  Med.  Jour.,  Jan.  20th, 
1917. 


THE   DESTRUCTION  OF  BACTERIA  97 

Practical  experience  seems  to  have  shown,  however,  that  the  healing 
of  wounds  that  have  been  treated  with  acriflavine  may  be  to  a  certain 
extent  delayed  by  the  action  of  the  dye,  which  is,  therefore,  not 
entirely  without  effect  on  tissue  cells.  Proflavine,  a  closely  related 
dye,  is  similar  in  its  effects  and  less  expensive  in  preparation. 

Triphenylmethane  Dyes. — Many  of  the  dyes  of  this  series,  notably 
gentian  violet,  malachite  green  and  brilliant  green,  have  long  been 
known  to  be  highly  toxic  for  bacteria. 

/C6H5 

C^C6H4=N(CH3)2-C1 
\C6H4-N(CH3)2 
Malachite  green. 

They  are  of  especial  interest  since  their  toxic  action  seems  to  be 
directed  almost  entirely  against  Gram-positive  bacteria,  and  some- 
times a  dilution  one  thousand  times  or  more  as  strong  as  is  necessary 
to  inhibit  the  growth  of  streptococci  and  staphylococci,  will  be 
needed  for  prevention  of  growth  of  the  Colon  bacillus.  Since  most 
of  the  common  pyogenic  organisms  are  Gram-positive,  the  use  of 
these  substances  in  wound  dressings  suggests  itself.  Practically, 
it  has  been  found,  particularly  with  brilliant  green,  that,  while  it 
acts  as  a  very  efficient  antiseptic  in  wounds,  here  also  the  dye  is 
not  without  effect  on  the  tissue  cells,  and  the  granulations  that  are 
formed  in  its  presence  are  not  of  the  normal  type.  Concentrated 
solutions  of  crystal  violet  and  brilliant  green  have  been  used  for 
sterilizing  the  skin  before  operation,  and  are  claimed  to  be  more 
efficient  for  this  purpose  than  tincture  of  iodin.52 

Methods  of  Testing  the  Efficiency  of  Disinfectants.— The  effi- 
ciency of  any  given  disinfectant  depends,  as  we  have  seen,  upon  a 
number  of  factors,  any  one  of  which,  if  variable,  may  lead  to  con- 
siderable differences  in  the  end  result.  Thus,  as  far  as  the  bacteria 
themselves  are  concerned,  it  is  necessary  to  remember  that  not  only 
do  separate  species  differ  in  their  resistance  to  disinfectants,  but 
that  different  strains  within  the  same  species  may  show  such  varia- 
tions as  well.  This  fact  largely  accounts  for  the  widely  varying 
reports  made  by  different  investigators  as  to  the  resistance  of 
anthrax  spores,  and  depends  possibly  upon  temporary  or  permanent 
biological  differences  produced  in  bacteria  by  the  conditions  of  their 
previous  environment. 


*2Bonney  and  Browning,  Brit.  Med.  Jour.,  May  18th,  1918. 


98  BIOLOGY  AND  TECHNIQUE 

The  numbers  of  bacteria  exposed  to  the  disinfectant,  further- 
more, is  a  factor  which  should  be  kept  constant  in  comparative 
tests.  The  medium,  moreover,  in  which  bacteria  are  brought  into 
contact  with  the  disinfectant  is  a  matter  of  great  importance,  inas- 
much as  either  by  entering  into  chemical  combination  with  the  dis- 
infectant it  may  detract  from  its  concentration  or  by  coagulation 
it  may  form  a  purely  mechanical  protection  for  the  microorganism. 
Thus  bacteria  which  may  be  destroyed  in  distilled  water  or  salt- 
solution  emulsion  with  comparative  ease,  may  evince  an  apparently 
higher  resistance  if  acted  upon  in  the  presence  of  blood  serum, 
mucus,  or  other  albuminous  substances.  Temperature  influences 
bactericidal  processes  in  that  most  chemical  disinfectants  are  more 
actively  bactericidal  at  higher  than  at  lower  temperatures,  a  fact 
due  most  likely  to  the  favorable  influence  of  temperature  upon  all 
chemical  reactions.53  As  far  as  merely  inhibitory  or  antiseptic 
values  are  concerned,  however,  the  temperature  least  favorable  for 
the  reaction  of  the  antiseptic  is  that  which  represents  the  optimum 
growth  temperature  for  the  microorganism  in  question  and  the  in- 
hibitory effects  of  any  substance  are  less  marked  at  this  point  than 
at  temperatures  above  or  below  it. 

The  important  influence  exerted  by  the  solvent  in  which  the 
disinfectant  is  employed  has  already  been  discussed.  For  ordinary 
work  it  is  customary  to  express  absolute  and  comparative  antiseptic 
and  bactericidal  values  in  terms  of  percentages  based  upon  weight, 
and  this,  beyond  question,  is  both  simple  and  practical.  For  strictly 
scientific  comparisons,  however,  as  Kronig  and  Paul54  have  pointed 
out,  it  is  by  far  more  accurate  to  work  with  equimolecular  solutions. 

Eideal  and  Walker55  have  devised  a  method  of  testing  disin- 
fectants, in  which  an  attempt  is  made  to'  establish  a  standard  for 
comparisons.  They  choose,  as  the  standard,  carbolic  acid,  and  es- 
tablish what  they  call  the  "carbolic-acid  coefficient,"  This  coeffi- 
cient they  obtain  in  the  following  way:  the  particular  dilution  of 
the  disinfectant  under  investigation  which  will  kill  in  a  given  time, 
is  divided  by  the  strength  of  carbolic  acid  which,  under  the  same 
conditions,  will  kill  the  same  bacteria  in  the  same  time.  We  quote 


63  v.  Behring,  "Bekaempf.  cler  Infektions-Krankh.,  Infektion  u.  Desinf ection, 
Leipzig,  1894. 

64  Kronig  und  Paul,  loc.  cit. 

65  Eideal  and  Walker,  Jour,  of  the  Sanitary  Ins.,  London,  xxiv. 


THE  DESTRUCTION  OF  BACTERIA 


99 


an  example  of  such  a  test,  given  by  Simpson  and  Hewlett,50  com- 
paring formalin  and  carbolic  acid. 

BACILLUS  PESTIS. 


Sample. 


Formalin .... 
Carbolic  acid 


Dilution. 

TIME  IN 

MINUTES. 

2.5 

5 

7.5 

10 

12.5 

15 

lin  30 
1  in  40 
1  in  100 

growth 
growth 

growth 
growth 

growth 

1  in  110 

growth 

growth 

In  the  above  table,  formalin  1  in  30  killed  in  the  same  time  as 
carbolic  acid  1  in  110.  Thus  the  carbolic-acid  coefficient  of  formalin 
in  this  test  =  3%10  =  .27. 

The  Rideal-Walker  method  has  been  much  used  and  is  recom- 
mended by  many  workers.57 

The  most  precise  method  of  standardizing  disinfectants  is  that 
now  in  use  in  the  U.  S.  Public  Health  Service.  It  is  a  modification 
of  the  Rideal-Walker  procedure  devised  by  Anderson  and  Mc- 
Clintic.58 

Stock  5  per  cent  solutions  of  the  disinfectant  in  question  and  of 
the  standard  (phenol)  are  first  prepared  and  a  series  of  accurate 
dilutions  made  with  distilled  water  using  graduated  pipettes.  (To 
make  1 :70  take  4  c.c.  of  stock  and  10  c.c.  distilled  water ;  1 :80  =  4 
c.c.  of  stock  +  12  c.c.  distilled  water ;  1 :90  .=  4  c.c.  stock  +  14  c.c. 
distilled  water;  1:500  =  2  c.c.  of  stock  +  48  c.c.  of  distilled  water. 
Complete  dilution  tables  are  given  in  their  original  article.)  The 
series  should  include  dilutions  strong  enough  to  kill  B.  typhosus 
in  two  and  a  half  minutes  and  weak  enough  to  fail  to  do  so  in 
fifteen  minutes.  If  dilutions  greater  than  1-500  are  required,  a 
second  1  per  cent  stock  solution  is  prepared.  They  adopted  the 
following  scale  for  their  tests :  Dilutions  up  to  1 :70  should  vary 
from  the  next  in  the  series  by  a  difference  of  5  (i.e.,  5  parts  of 
water),  and  so  on  if  higher  solutions  are  necessary. 


"  Simpson  and  Hewlett,  Lancet,  ii,  1904. 

57  Sommerville,  Brit.  Med.  Jour.,  1904. 

68  Anderson  and  McClintic,  Jour,  of  Inf.  Dig.,  1911,  viii,  1, 


100  BIOLOGY  AND  TECHNIQUE 

From  1:70       to  1:160     by  a  difference  of  10 

From  1:160     to  1:200     by  a  difference  of  20 

From  1:200     to  1:400     by  a  difference  of  25 

From  1:400     to  1:900     by  a,  difference  of  50 

From  1:900     to  1:1800  by  a  difference  of  100 

From  1:1800  to  1:3200  by  a  difference  of  200 

Short  wide  test  tubes  1  inch  by  3  inches  are  used  in  making  the 
test.  These  are  placed  in  a  rack  in  a  water  bath  at  20°  C.  Five 
c.c.  of  each  dilution  are  measured  into  a  series  of  these  tubes  begin- 
ning with  the  strongest  specimen  and  rinsing  the  pipette  once  with 
each  dilution  before  the  5  c.c.  are  measured  out.  For  inoculation, 
a  24-hour  broth  culture  of  B.  typhosus  is  prepared  which  has  been 
transferred  daily  for  at  least  3  days.  Before  use  it  is  shaken  and 
filtered  through  sterile  filter  paper.  The  wide  test  tubes  containing 
diluted  disinfectant  are  inoculated  with  1/10  c.c.  of  this  culture 
with  a  graduated  pipette.  The  tip  of  the  pipette  is  held  against 
the  side  of  the  tube  to  insure  accurate  measurement  and  the  tube 
immediately  shaken  to  mix  the  bacteria  thoroughly  with  the  dis- 
infectant. Test  inoculations  are  made  from  this  mixture  at  proper 
intervals  into  tubes  containing  10  c.c.  of  standard  extract  broth 
of  +1.5  acidity,  using  loops  4  mm.  in  diameter.  At  least  four 
such  loops  should  be  at  hand,  supported  on  a  rack  or  wooden  block 
so  that  a  fan-tail  Bunsen  burner  may  be  placed  under  each  wire 
in  turn.  Each  one  is  sterilized  after  a  plant  is  made  and  allowed 
to  cool  while  the  other  three  are  being  used  in  order. 

The  test  is  conducted  as  follows:  A  row  of  ten  wide  tubes  con- 
taining dilutions  of  the  antiseptic  is  placed  in  the  water  bath  at 
20°  C.  and  time  allowed  for  them  to  reach  the  temperature  of  the 
bath.  They  are  then  inoculated  in  order  at  intervals  of  exactly 
15  seconds.  Fifteen  seconds  after  the  last  tube  has  been  inoculated 
a  subculture  is  made  from  the  first  tube  of  the  series  (i.e.,  2y2 
minutes  after  this  first  tube  was  inoculated)  and  from  the  other 
tubes  in  order  at  15-second  intervals.  Fifteen  seconds  after  this 
first  series  of  subcultures  is  completed,  a  second  series  of  subcultures 
is  begun  which  will  give  the  result  of  a  5-minute  exposure  to  the 
antiseptic  and  the  subinoculations  continued  at  15-second  intervals 
until  all  dilutions  have  been  tested  for  fifteen  minutes.  If  the 
strength  of  the  antiseptic  is  known  approximately,  subcultures  of 
the  lower  dilutions  for  the  longer  periods  may  be  omitted.  It  is 
convenient  to  have  an  assistant  at  hand  to  call  time  and  to  label 


THE   DESTRUCTION  OF  BACTERL*  M31 

the  subcultures  as  soon  as  made.  The  tubes  may,  however,  be  placed 
in  order  in  suitable  racks  without  labeling.  The  subculture  tubes 
are  incubated  for  48  hours  at  37°  C.  and  those  in  which  growth  is 
observed  are  recorded  positive. 


DETERMINATION  OF  THE  CARBOLIC-ACID  COEFFICIENT 
OF  A  DISINFECTANT. 

(ANDERSON  AND  McCLiNTic) 

NAME "A" 

TEMPERATURE  OF  MEDICATION 20°  C. 

CULTURE  USED  B.  TYPHOSUS 24-hr!,  Extract  Broth,  Filtered 

PROPORTION  OF  CULTURE  AND  DISINFECTANT 0.1  c.c. +5  c.c. 

ORGANIC  MATTER,  NONE;     KIND,  None;     AMOUNT,  None. 

SUBCULTURE   MEDIA Standard  Extract  Broth 

REACTION : +1.5 

QUANTITY  IN  EACH  TUBE 10  c.c. 


Sample. 

Dilution. 

Time  Culture  Exposed  to  Action 
of  Disinfectant  for  Minutes. 

Phenol  Coefficient. 

VA 

5 

71A 

10 

12K 

15 

Phenol  

1      80 

— 

— 



1      90 

+ 

— 

— 

— 

80)375 

1     100 

+ 

+ 

+ 

— 

— 

— 

4.69 

1     110 

+ 

+ 

+ 

+ 

+ 

— 

110)650 

5.91 

Disinfectant  "A"... 

1     350 
1     375 

: 

: 

: 

2)10.60 

1     400 

+ 

— 

— 

— 

5.30  = 

1     425 

+ 

+ 

— 

— 

— 

— 

coefficient 

1     450 

+ 

+ 

— 

— 

— 

— 

1     500 

+ 

+ 

— 

— 

— 

— 

1     550 

+ 

+ 

+ 

— 

- 

- 

1     600 

+ 

+ 

+ 

4- 

— 

— 

1     650 

+ 

+ 

+ 

+ 

+ 

— 

1     700 

+ 

+ 

+ 

+ 

+ 

+ 

1     750 

+ 

+ 

+ 

4- 

+ 

+ 

To  obtain  the  coefficient  the  weakest  dilution  of  the  unknown 
antiseptic  which  kill  in  2y2  minutes  is  divided  by  the  weakest 
dilution  of  phenol  which  kills  in  the  same  time.  The  same  is  done 
for  the  weakest  strength  that  kills  in  15  minutes  and  an  average  is 
taken.  The  results  of  such  a  test  are  shown  in  the  table  on  page  104. 


}02  BIOLOGY  AND  TECHNIQUE 

As  only  the  2i/2-imnute  and  15-minute  intervals  are  used  in 
determining  this  result  it  seems  unnecessary  to  make  plants  at  .the 
intervening  periods  except  in  special  cases  where  more  detailed 
information  is  desired. 

The  procedure  may  be  modified  by  adding  some  organic  sub- 
stance such  as  killed  bacteria  to  the  diluted  antiseptic.  For  many 
substances,  e.g.,  bichloride  of  mercury,  the  antiseptic  value  in 
presence  of  organic  matter  is  much  lower  than  in  watery  solution. 
Anderson  and  McClintic  insist  that  great  care  in  making  the  dilu- 
tions and  rigid  adherence  to  a  uniform  technique  are  necessary  to 
obtain  consistent  results  in  such  tests. 

DETERMINATION  OF  ANTISEPTIC  VALUES. — The  antiseptic  or  in- 
hibitive  strength  of  a  chemical  substance,  sometimes  spoken  of  as 
the  "coefficient  of  inhibition,"  is  determined  by  adding  to  definite 
quantities  of  a  given  culture  medium,  graded  percentages  of  the 
chemical  substance  which  is  being  investigated,  and  planting  in  these 
mixtures  equal  quantities  of  the  bacteria  in  question.  The  medium 
used  for  the  tests  may  be  nutrient  broth  or  melted  gelatin  or  agar. 
If  broth  is  used,  growth  is  estimated  by  turbidity  of  the  medium 
and  by  morphological  examination ;  if  the  agar  or  gelatin  is  em- 
ployed, plates  may  be  poured  and  actual  growth  observed. 

Thus,  in  the  case  of  carbolic  acid,  a  5  or  10  per  cent  solution  is 
prepared  and  added  to  tubes  of  the  medium,  as  follows: 

Tube  1  contains  5%  carbolic  2  c.c.  -f-  broth  8  c.c.  —  1:1,000  carbolic  acid. 

"  2        "       •  5            "  1  c.c.  -f  broth  9  c.c.  =  1:200           "  " 

"  3  5            "  .5  c.c.  -f  broth  9.5  c.c.  —  1:400           "  " 

"  4        "  5            "  7  c.c.  4-  broth  9.8  c.c.  =  1:1,000        "  «« 

"  5        "  5            "  .1  c.c.  -f  broth  9.9  c.c.  =  1:1,500        "  " 

To  each  of  these  tubes  a  definite  quantity  of  the  bacteria  is  added 
either  by  means  of  a  standard  loopful  of  a  fresh  agar  culture,  or 
better  by  a  measured  volume  of  an  even  emulsion  in  sterile  salt 
solution.  The  inoculated  tubes  are  then  incubated  at  a  temperature 
corresponding  to  the  optimum  growth  temperature  for  the  micro- 
organism in  question.  The  tubes  are  examined  for  growth  from 
day  to  day.  From  tubes  containing  higher  dilutions,  in  which  no 
growth  is  visible,  transplants  are  made  to  determine  the  presence 
of  living  bacteria  and  to  distinguish  between  inhibition  or  antisepsis 
and  bacterial  death  or  disinfection. 


THE  DESTRUCTION  OF  BACTERIA 


103 


INHIBITION  STRENGTHS  OF  VARIOUS  ANTISEPTICS. 
ADAPTED  FROM  FLUGGE,  LEIPZIG,  1902. 


• 

Anthrax  Bacilli. 

Other  Bacteria. 

Putrefactive  Bac- 
teria in  Bouillon. 

ACIDS 

Sulphuric     .    

1  :  3,000 

Choi.  spir.  1  :  6,000 

Hydrochloric       

1  :  3,000 

B  diph  1  :  3,000 

Sulphurous 

B.  mallei  1  :  700 
B.  typh.  1  :  500 
Choi  spir  1  :  1,000 

1  :  6,000 

Arsenous 

1  •  200 

Boric                

1  :  800 

. 

1  :  100 

ALKALIES 
Potass,  hydroxid  

Ammon.  hydroxid  
Calcium  hydroxid  

SALTS 
Copper  sulphate 

1  :  700 
1  :  700 

B.  diphth.  1  :  600 
Choi.  spir.  1  :  400 
B.  typh.  1  :  400 
Choi.  spir.  1  :  500 
B.  typh.  1  :  500 
Choi.  spir.  1  :  1,100 
B.  typh.  1  :  1,100 

1  :  1,000 

Ferric  sulphate 

1  :  90 

Mercuric  chlorid  
Silver  nitrate 

1     100,000 
1     60,000 

B.  typhosus  1  :  60,000 
Choi  spir 

1  :  20,000 

Potass  perman 

1     1  000 

B.  typhosus  1  :  50,000 

1     500 

HALOGENS  AND  COMPOUNDS 
Chlorin 

1     1  500 

1     4,000 

Bromin 

1     1  500 

1     2,000 

lodin 

1     5000 

1     5,000 

Potass  iodid 

1     7 

ORGANIC  COMPOUNDS 
Ethvl  alcohol 

1  •  12 

• 

1     10 

Acetic  and  oxalic  acids  .... 
Carbolic  acid  

Benzoic  acid.  .  

1  :  800 
1  :  1,000 

B.  diph.  1  :  500 
B.  typh.  1  :  400 
Choi.  spir.  1  :  600 

1     400 

Salicylic  acid  
Formalin     (4%    formaldo- 
hycl)    

1  :  1,500 

Choi.  spir.  1  :  20.000 

1  :  1,000 

Camphor  

1     1,000 

Staphylo.  1  :  5,000 

Thymol 

1     10  000 

1  :  3,500 

Oil  mentha  pip  

1     3,000 

Oil  of  terebinth  
Peroxide  of  hydrogen  

1     8,000 



1  :  2,000 

104 


BIOLOGY  AND  TECHNIQUE 


BACTERICIDAL  STRENGTHS  OF  COMMON  DISINFECTANTS. 
ADAPTED  FROM  FLUGGE,  LEIPZIG,  1902. 


Strepto-  and 
Staphylo- 
cocci. 

Anthrax  and  Typhoid  Bacilli. 
Cholera  Spirillum. 

Anthrax  Spores. 

5  Minutes. 

5  Minutes. 

2-24  Hours. 

ACIDS 
Sulphuric 

1  :  10 
1  :10 

1  :  100 
1  :  100 

1  :  1,500 
1  :  1,500 
(Typhoid 
1  :  700) 
1  :  300  (Gas 
10vol.  %) 
1  :  30 

1  :  1,000 

1  :  10,000 
1  :  4,000 

1  :  50  in  10  days 
1  :  50  in  10  days 

Cone.  sol.  incom- 
plete disinfection 

1  :  20  (5  days) 
1  :  2,000  (26  hours) 

1  :  20  (1  day) 
1  :20  (1  hour) 

2  per  cent  (in  1  hr.) 
1  :  1,000  (in  12  hrs.) 

Alcol.    50%    for    4 
months    without 
killing    spores. 
Koch.59 
1  :  20  (4-45  days) 
(at  40°  in  3  hrs.) 

(10%  in  5  hrs.) 

1     20  (in  6  hrs.) 
1     100  (in  1  hr). 
3     100  (in  1  hr.) 

Hydrochloric  
Sulphurous          

Sulphurous  

Boric 

ALKALIES 
Potass,  hydroxid  
Ammon    hvdroxid   . 

1  :  5 

1  :  300 
1  :  300 

1  :  2,000 

Calcium  hydroxid.  .  .  . 

SALTS 
Copper  sulphate  
Mercuric  chlor  

Silver  nitrate 

1  :  10,000  to 
1,000 
1  :  1,000 
1  :  200 

1  per  cent 
1  :200 

70%-15  mins. 

1  :  60 
1  :300 

Potass  perrnang  

Chlorinated  lime  

HALOGENS   AND   COM- 
POUNDS 
Chlorin  
Trichlorid  of  iodin  .... 

ORGANIC  COMPOUNDS 
Ethyl  alcohol  
Acetic  and  oxalic  acids. 

Carbolic  acid  
Lysol      

1  :  500 

.  1  per  cent 
1  :  1,000 

70%-lOmins. 

Cholera  1  :200 
Typh.  1  :  50 
1  :  300 
1  :  100 

1  :  20 
1  :  200 

1  :  2-300 

1  :  300 
1  :  3,000 

1  :  1,000 
1  :  500 

Creolin 

Salicylic  acid  
Formalin  (40%  formal- 
dehyde)   

1  :  1,000 

1  :  10 
Cone. 

Peroxide  of  hydrogen.  . 

59  Koch,  Arb.  a.  d.  kais.  Gesundheitsamt,  1,  1881 


THE   DESTRUCTION   OF  BACTERIA  105 

The  determination  of  the  bactericidal  or  disinfectant  value  of 
a  chemical  substance  upon  spores  may  be  carried  out  by  a  variety 
of  methods.  Koch,60  using  anthrax  spores  as  the  indicator,  dried 
the  spores  upon  previously  sterilized  threads  of  silk.  These  were 
exposed  to  the  disinfectant  at  a  definite  temperature  for  varying 
times,  the  disinfectant  was  then  removed  by  washing  in  sterile  water, 
and  the  threads  planted  upon  gelatin  or  blood  serum  media  and 
incubated.  A  serious  objection  to  this  method  was  pointed  out  by 
Geppert,61  who  maintains  that  it  is  impossible  by  simple  washing 
to  remove  completely  the  disinfectant  in  which  the  thread  has  been 
soaked.  This  author  suggests  that,  whenever  possible,  the  disin- 
fectant, at  the  end  of  the  time  of  exposure,  should  be  removed  by 
chemical  means.  In  the  case  of  bichloride  of  mercury,  Geppert  ex- 
poses emulsions  of  the  bacteria  to  aqueous  solutions  of  the  disin- 
fectant, and  at  the  end  of  exposure  precipitates  out  the  bichloride 
of  mercury  with  ammonium  sulphide.  In  the  case  of  a  large  number 
of  disinfectants,  however,  this  is  not  possible,  and,  when  the  thread 
method  is  used,  removal  of  the  chemical  agent  by  washing  must  be 
practiced.  Complete  removal  of  the  disinfectant  is  especially  de- 
sirable, since  spores  previously  exposed  to  these  substances  are  more 
easily  inhibited  by  dilute  solutions  than  are  normal  spores.  The 
spores  may  be  dried  upon  the  end  of  a  glass  rod,  which,  after 
exposure,  is  washed  in  distilled  water  or  salt  solution  and  then 
immersed  in  sterile  broth.62 

A  simple  method  is  that  in  which  graded  percentages  of  the 
disinfectant  are  added  to  the  menstruum,  blood,  blood  serum,  broth, 
etc.,  in  which  the  disinfectant  is  to  be  tested,  and  equal  quantities 
of  bacteria  thoroughly  emulsified  in  water  or  salt  solution  are  added. 
Loopfuls  of  these  mixtures  are  then  planted  from  time  to  time  in 
agar  or  gelatin  plates  upon  which  colony  counts  can  afterward  be 
made. 

In  all  such  tests,  it  is  important  to  remember  that  the  presence 
of  organic  fluids,  blood  serum,  mucus,  etc.,  considerably  alters  the 
efficiency  of  germicides,  and  whenever  practical  deductions  are  made, 
experimental  imitation  of  the  actual  conditions  should  be  attempted. 

Gaseous  Disinfectants  for  Purposes  of  Fumigation. — There  are  a 
large  number  of  gaseous  agents  which  are  harmful  to  bacteria.  Only 

80  Koch,  Arb.  a.  d.  kais.  Gesundheitsamt,  1,  1881. 

61  Geppert,  Berl.  klin.  Woch.,  xxvi,  1889. 

"Hill,  Rep.  Am.  Pub.  Health  Assn.,  xxiv,  1898. 


106  BIOLOGY  AND  TECHNIQUE 

a  few,  however,  are  of  sufficient  bactericidal  strength  to  be  of  prac- 
tical importance. 

Oxygen,  especially  in  the  nascent  state,  may  exert  distinct  bac- 
tericidal action  upon  some  bacteria.  That  strictly  anaerobic  strains 
are  inhibited  by  its  presence  has  already  been  mentioned. 

Ozone  was  shoAvn  by  Ransome  and  Fullerton63  to  exert  consider- 
able germicidal  power  when  passed  through  a  liquid  medium  in 
which  bacteria  were  suspended,  but  was  absolutely  without  activity 
when  employed  in  the  dry  state. 

Chlorine  because  of  its  powerful  germicidal  action  was  once 
looked  upon  with  favor,  but  has  been  found  quite  inadequate  from 
a  practical  point  of  view  because  of  its  injurious  action  upon  ma- 
terials, and  its  irregularity  of  action.  Chlorine,  too,  is  but  weakly 
efficient  unless  in  the  presence  of  moisture.64 

Sulphur  dioxide  or  sulphurous  anhydrid  (S02),  formerly  much 
used  for  room  disinfection,  is  no  longer  regarded  as  uniformly  effi- 
cient for  general  use.  The  gas  is  produced  by  burning  ordinary  roll 
sulphur,  conveniently  in  a  Dutch  oven.  To  be  at  all  effective,  water 
should  be  vaporized  at  the  same  time,  since  the  disinfectant  action 
is  dependent  upon  the  formation  of  sulphurous  acid.  The  concen- 
tration of  the  gas  should  be  at  least  8  per  cent  of  the  volume  of 
air  in  the  room.  For  this  purpose  about  three  pounds  of  sulphur 
should  be  burned  for  every  thousand  cubic  feet  of  space.  It  should 
be  allowed  to  act  for  not  less  than  twenty-four  hours.  The  researches 
both  of  Wolff hugel65  and  of  Koch66  have  'shown  that  the  gas  is  not 
sufficient  for  the  destruction  of  spores.  Park67  believes  that  sulphur 
dioxid  used  in  quantities  of  four  pounds  of  sulphur  to  1,000  cubic 
feet  is  of  practical  value  for  fumigating  purposes  in  cases  of  diph- 
theria and  the  exanthemata.  Sulphur  dioxid  fumigation  is  more 
effective  than  formaldehyd  for  the  destruction  of  insects — fleas,  lice 
and  bedbugs — a  matter  of  importance  in  epidemics  of  typhus  fever, 
relapsing  fever,  plague,  etc. 

Of  all  known  gaseous  disinfectants  by  far  the  most  reliable  is 
formaldehyd.  In  all  cases  where  formaldehyd  fumigation  is  in- 


*3Bansome  and  Fullerton,  Eep.  Public  Health,  July,  1901. 

"Fischer  and  Proskauer,  Mitt.  a.  d.  kais.  Gesundheitsamt,  x,  11,  1882. 

65  Wol/fhugel,  Mitt.  a.  d.  kais.  Gesundheitsamt,  i,  1881. 

66  Koch,  Mitt.  a.  d.  kais.  Gesundheitsamt,  i,  1881. 

67  Park,  "Pathogen.  Bact.,"  N.  Y.,  1908. 


THE   DESTRUCTION   OF   BACTERIA  107 

tended,  clothing,  bed-linen,  and  fabrics  should  be  spread  out,  cup- 
boards and  drawers  freely  opened.  The  cracks  of  windows  and 
doors  should  be  hermetically  sealed  with  paper  strips  or  by  calking 
with  cotton.  In  all  cases  moisture  should  be  provided  for,  either 
in  the  generating  apparatus  or  by  a  separate  boiler. 

Direct  evaporation  of  formaldehyd  from  formalin  solutions  has 
been  the  principle  underlying  most  of  the  methods.  If  such  evapora- 
tion is  attempted  from  an  open  vessel,  however,  polymerization  of 
formaldehyd  to  the  solid  trioxymethylene  occurs.  To  prevent  this, 
Trillat68  and  others  have  constructed  special  autoclaves  in  which  20  per 
cent  of  calcium  chlorid  is  added  to  formalin  which  is  then  vaporized 
under  pressure. 

The  Trillat  autoclave,  as  well  as  others  constructed  on  the  same  principle, 
consists  of  a  strong  copper  chamber  of  a  capacity  of  about  a  gallon,  fitted 
with  a  cover  which  can  be  tightly  screwed  into  place.  The  cover  is  perforated 
by  an  outlet  vent,  a  pressure  gauge,  and  a  thermometer.  The  whole  apparatus 
is  adjusted  upon  a  stand  and  set  over  a  kerosene  lamp.  Into  the  chamber 
is  put  about  one-half  to  three-quarters  its  capacity  of  40  per  cent  formaldehyd 
(commercial  formalin)  containing  15-20  per  cent  calcium  chlorid.  The  solu- 
tion used  should  be  free  from  methyl  alcohol,  since  this  leads  to  the  formation, 
with  formaldehyd,  of  methylal,  which  is  absolutely  without  germicidal  action, 
for  a  room  of  about  3,000  cubic  feet  Trillat  advises  the  continuance  of  the 
gas  flow  for  about  an  hour.  The  method  is  not  uniformly  reliable. 

A  method  which  has  found  much  favor  is  that  in  which  glycerin 
—usually  in  a  concentration  of  10  per  cent — is  added  to  formalin. 
According  to  Schlossmann69  the  presence  of  glycerin  hinders  poly- 
merization. An  apparatus  in  which  this  mixture  is  conveniently 
utilized  is  that  of  Lentz.  Formalin  with  10  per  cent  glycerin  is 
placed  in  the  copper  tank  and  heated  by  a  burner.  This  apparatus 
has  been  favorably  endorsed  by  the  War  Department  of  the  United 
States. 

The  so-called  Breslau  method  of  generating  formaldehyd  depends 
upon  the  evaporation  of  formaldehyd  from  dilute  solutions,  v. 
Brunn70  claims  that  where  formalin  in  30  to  40  per  cent  strength 
is  evaporated,  water  vapor  is  generated  more  rapidly  than  formal- 
dehyd is  liberated,  and  a  concentration  leading  to  polymerization 


*  Trillat.  Compt.  rend,  de  1'acad.  dcs  sc.,  1896. 
99  Schlossmann,  Munch,  med.  Woch.,  45,  1898 
70  v.  Brunn,  Zeit.  f .  Hyg.,  xxx,  1899. 


108  BIOLOGY  AND  TECHNIQUE 

occurs.  If,  however,  dilution  is  carried  out  until  the  formaldehyd 
in  the  solution  is  not  more  than  8  per  cent,  the  generation  of  water 
vapor  and  formaldehyd  take  place  at  about  equal  speed  and  no 
concentration  occurs.  Schlossmann69  furthermore  claims  that  poly- 
merization in  the  vaporized  formaldehyd  does  not  occur  if  sufficient 
water  vapor  is  present — a  principle  which  may  also  contribute  to 
the  efficiency  of  the  Breslau  method. 

In  practice,  the  apparatus  devised  by  v.  Brunn  (Fig.  13)  consists  of  a 
strong  copper  kettle  of  about  34  cm.  diameter  by  7.5  cm.  height.  This  is 
completely  closed  except  for  two  openings  in  the  slightly  domed  top,  one 
of  which  is  the  exit  vent,  the  other,  laterally  placed,  is  for  purposes  of  filling 
and  is  closed  by  a  screw  stopper.  The  tank  is  filled  with  a  solution  of 
formalin  of  a  strength  of  from  8  to  10  per  cent  (commercial  formalin  1:4). 
The  apparatus  permits  the  evaporation  of  large  quantities  of  fluid  in  a  short 
time  (3  liters  in  one  hour).  When  the  lamp  is  left  in  a  closed  room  care 
should  be  taken  to  fill  it  with  a  quantity  of  alcohol  proportionate  to  the 
amount  of  fluid  to  be  evaporated.  This,  according  to  v.  Brunn,  is  about 
one-quarter  of  the  volume  of  formalin  solution  used.  By  using  1.5  liters 
of  8  per  cent  formalin  for  each  1,000  cubic  feet  of  space,  this  apparatus 
is  said  to  yield  a  concentration  of  formaldehyd  of  about  25  grams  to  the 
cubic  meter. 

To  do  away  with  the  use  of  liquid,  a  method  has  been  devised 
which  depends  on  principle  upon  the  breaking  up  by  heat  of  the 
solid  polymer  of  fortnaldehyd  (trixymethylene). 

The  apparatus  (trade  name,  "^chering's  Paraform  Lamp")  as  described 
by  Aronson71  consists  of  a  cylindrical  mantle  of  sheet-iron  placed  upon  a 
stand  and  supplied  below  with  an  alcohol  lamp.  Set  into  the  top  of  the 
mantle  is  a  small  chamber,  into  which  1  gram  tablets  of  trioxymethylene 
are  placed.  The  alcohol  lamp,  so  placed  that  the  wicks  project  but  slightly— 
to  avoid  overheating — is  lighted,  and  the  formalin  generated  passes  out 
through  slits  in  the  upper  case,  mingling  with  the  water  vapor  and  other 
gases  liberated  by  the  alcohol  flame.  The  more  modern  devices  have  water- 
boiler  attachments  to  insure  sufficient  moisture.  Two  tablets  are  sufficient 
for  the  fumigation  of  about  thirty-five  cubic  feet,  and  2  c.c.  of  alcohol 
are  filled  into  the  lamp  for  each  tablet.  One  hundred  to  one  hundred  and 
fifty  tablets  are  usually  enough  for  the  ordinary  room. 

A  simple  method  of  generating  formaldehyd  is  that  which  is 
known  as  the  "lime  method/'  In  a  wide  shallow  pan  40, per  cent 

"Aronson,  Zeit.  f.  Hyg.,  xxv,  1897. 


THE   DESTRUCTION   OF  BACTERIA  109 

formaldehyd  solution  (commercial  formalin)  is  poured  over  quick- 
lime (CaO).  The  previous  addition  of  concentrated  sulphuric  acid 
to  the  formalin,  in  proportions  of  one  to  ten,  increases  the  speed  of 
formalin  liberation,  and  aids  in  limiting  polymerization.  One  and 
one-half  to  two  pounds  (one-half  to  one  kilogram)  of  quick-lime 
are  used  for  every  500  c.c.  of  the  formalin  solution.  The  heat 
generated  in  the  slaking  of  the  lime  produces  volatilization  of  the 
formalin. 

A  modification  of  this  method  is  that  of  Schering72  in  which 
tablets  of  paraform  and  unslaked  lime  are  together  laid  into  a  pan 
and  warm  water  is  poured  over  them. 

A  very  simple  method  is  the  potassium  permanganate  method  of 
Evans  and  Russell.73  This  method  depends  upon  the  active  reaction 
occurring  when  formalin  and  potassium  permanganate  are  mixed. 
Permanganate  is  placed  into  a  bucket  and  the  formalin  poured  over 
it.  The  bucket  should  be  large  enough  to  prevent  overflowing  when 
the  mixture  foams.  Special  galvanized  iron  pails  are  made  with 
funnel-shaped  tops,  and  pails  should  be  placed  on  a  piece  of  iron 
sheeting  or  bricks  to  prevent  overheating  of  the  floor;  500  c.c.  of 
commercial  formalin  and  250  grams  of  potassium  per  manganate 
should  be  used  for  every  1,000  cu.  ft. 

The  room  in  which  formaldehyd  has  been  liberated  is  kept  sealed, 
in  the  manner  already  described,  for  at  least  twelve  hours,  after 
which  the  windows  and  doors  are  opened.  The  odor  which  remains 
after  formaldehyd  fumigation  may  be  removed  by  sprinkling  with 
ammonia,  or  by  the  use  of  anyone  of  the  various  sorts  of  apparatus 
devised  for  the  liberation  of  ammonia. 

For  the  destruction  of  rodents,  hydrocyanic  acid  gas  is  used  in* 
the  fumigation  of  ships  and  houses.  This  is  of  especial  importance 
in  controlling  such  diseases  as  plague.  Recently  Creel,  Faget,  and 
Wrightson,  of  the  United  States  Public  Health  Service,  have  studied 
this  method.  They  found  hydrocyanic  acid  gas  is  more  penetrating, 
more  toxic,  and  more  easily  applied  than  either  sulphur  dioxid  or 
carbon  monoxid.  5  oz.  of  powdered  potassium  cyanid  per  1,000 
cu.  ft.  of  space  were  sufficient  to  kill  rodents.  The  gas  is  produced 
by  dropping  the  potassium  cyanid  into  sulphuric  acid  of  a  specific 
gravity  of  1.84,  or  commercial  grade  66B.  The  gas  is  as  effective 


72  Schering,  Hyg.  Rundschau,  1900. 

n  Evans  and  Russell,  Rep.  State  Bd.  Health,  Maine,  1904. 


110  BIOLOGY  AND  TECHNIQUE 

for  insects  as  it  is  for  rodents.  The  element  of  danger  to  human 
life  must  be  always  considered  in  carrying  out  such  fumigation,  but 
the  writers  referred  to  believe  that  there  is  no  danger  to  men  enter- 
ing a  place  fumigated  in  this  way  30  minutes  or  longer  after  the 
apertures  have  been  opened.  Subsequent  experience  -in  the  fumiga- 
tion of  vessels  has  shown  that  longer  periods  of  ventilation  are 
necessary,  depending  on  the  conditions  of  moisture  and  air  currents. 
In  spite  of  rigorous  precautions,  loss  of  life  has  occasionally  occurred 
when  cyanide  is  used  as  a  routine  procedure.  For  the  fumigation 
of  the  hold  of  a  ship,  Creel,  Faget,  and  Wrightson74  use  an  ordinary 
wooden  barrel,  into  the  top  of  which  is  placed  a  large  galvanized 
iron  funnel.  The  cyanide,  in  5-gal.  tins  with  top  removed,  may  be 
attached  to  the  funnel  and  dumped  into  the  sulphuric  acid  by  means 
of  a  rope  attached  to  the  tin,  after  the  barrel  has  been  lowered. 


74  Creel,  Faget,  and  Wrightson,  United  States  Public  Health  Keports,  Vol.  30, 
No.  39,  Dec.  3,  1915. 


CHAPTER   VI 

METHODS    USED    IN    THE    MICEOSCOP1C    STUDY    AND    STAINING    OF 

BACTERIA 

MICROSCOPIC    STUDY    OF    BACTERIA 

BACTERIA  may  be  studied  microscopically,  in  the  living  and  un- 
stained state,  and,  after  the  application  of  dyes,  in  colored  prepara- 
tions. For  the  manipulation  of  bacteria  for  such  study,  glass  slides 
and  coverslips  of  various  design  are  used.  These  must  be  perfectly 
clean  if  the  preparations  are  to  be  of  any  value.1 

The  Study  of  Bacteria  in  the  Living  State. — Living  bacteria  may 
be  studied  in  what  is  spoken  of  as  the  " hanging-drop"  preparation. 
For  this  purpose  a  so-called  hollow  slide  is  employed,  in  the  center 
of  which  there  is  a  circular  concavity  about  three-quarters  of  a 
centimeter  to  one  centimeter  in  diameter.  The  preparation  is 
manipulated  as  follows:  if  the  bacteria  are  growing  in  a  fluid 
medium,  a  drop  of  the  culture  fluid  is  transferred  to  the  center  of 
a  cover-slip.  If  taken  from  solid  media,  an  emulsion  may  be  made 
in  broth  or  in  physiological  salt  solution,  and  a  drop  of  this  trans- 
ferred to  the  cover-slip,  or  the  bacteria  may  be  emulsified  in  a  drop 
of  salt  solution,  or  broth,  directly  upon  the  cover-slip.  The  con- 
cavity on  the  slide,  having  first  been  rimmed  with  vaseline,  by 
means  of  a  small  camel's-hair  brush,  the  cover-slip  is  inverted  over 


1  Although  the  silicates  of  which  glass  is  composed  are  extremely  stable,  never- 
theless alkaline  silicates  which  are  said  to  separate  out  on  the  surface,  together 
with  grease  and  dirt  left  upon  the  glass  by  handling,  during  blowing  and  cutting, 
necessitate  cleansing  before  use.  This  may  be  accomplished  by  a  variety  of 
methods.  A  simple  one  suitable  for  general  application  is  as  follows:  (1)  The 
slides  and  coverslips  are  thrown  singly  into  boiling  water  and  left  there  for 
half  an  hour;  (2)  wash  in  twenty-five  per  cent  sulphuric  acid;  (3)  rinse  in 
distilled  water;  (4)  wash  in  alcohol;  (5)  wipe  with  a  clean  cloth  and  keep 
dry  under  a  bell-jar.  Another  method  convenient  for  routine  use  is  to  immerse, 
after  thorough  washing  in  soap-suds  and  acid,  in  ninety-five  per  cent  alcohol  and 
to  leave  in  this  until  the  time  of  use. 

Ill 


112  BIOLOGY  AND  TECHNIQUE 

the  slide  in  such  a  way  that  the  drop  hangs  freely  within  the  hollow 
space.  The  preparation  is  then  ready  for  examination  under  the 
microscope. 

Another  method,  known  as  the  "hanging  block  method,"  devised 
by  Hill,2  for  the  study  of  living  bacteria  in  solid  media  is  carried 
out  as  follows :  nutrient  agar  is  poured  into  a  Petri  dish  and  allowed 
to  solidify.  Out  of  this  layer  a  piece  about  a  quarter  of  an  inch 
square  is  cut.  This  is  placed  on  a  sterile  slide.  The  upper  surface 
of  the  agar  block  is  then  inoculated  with  bacteria  by  surface  smear- 
ing, and  the  preparation  covered  with  a  sterile  dish  and  allowed 
to  dry  for  a  few  minutes  in  the  incubator.  A  sterile  cover-slip  is 
then  dropped  upon  the  surface  of  the  block  and  sealed  about  the 
edges  with  agar.  Block  and  cover-slip  are  then  taken  from  the 
slide  and  fastened  over  a  moist  chamber  with  paraffin.  The  entire 
preparation  can  be  placed  upon  the  stage  of  a  microscope.  This 
method  is  especially  designed  for  the  study  of  cell-division. 


FIG.  9. — HANGING-DROP  PREPARATION. 

Living  bacteria  may  also  be  studied  in  stained  preparations  by 
the  so-called  " intra vital' '  method  of  Nakanishi.  Thoroughly 
cleaned  slides  are  covered  with  a  saturated  aqueous  solution  of 
methylene-blue.  This  is  spread  over  the  slide  in  an  even  film  and 
allowed  to  dry.  After  drying  the  slide  should  appear  of  a  trans- 
parent sky-blue  color.  The  microorganisms  which  are  to  be  examined 
are  then  emulsified  in  water,  or  are  taken  from  a  fluid  medium  and 
placed  upon  a  cover-slip.  This  is  dropped,  face  downward,  upon 
the  blue  ground  of  the  slide.  In  this  way  bacteria  may  be  stained 
without  being  subjected  to  the  often  destructive  processes  of  heat 
or  chemical  fixation.  According  to  Nakanishi,  cytoplasm  is  stained 
blue,  while  nuclear  material  assumes  a  reddish  or  purplish  hue. 

The  Study  of  Bacteria  in  Fixed  Preparations, — Stained  prepara- 
tions of  bacteria  are  best  prepared  upon  cover-slips,  the  process 
consisting  of  the  following  steps:  (1)  Spreading  on  cover-slip;  (2) 
drying  in  air;  (3)  fixing;  (4)  staining;  (5)  washing  in  water;  (6) 
blotting;  (7)  mounting. 


2  Hill,  Jour,  of  Med.  Eeseareh,  vii,  1902. 


MICROSCOPIC   STUDY   AND  STAINING  113 

(1)  Smearing. — Bacteria  from  a  fluid  medium  are  transferred  in 
a  small  drop  of  the  fluid,  with  a  platinum  loop,  to  a  cover-slip  and 
carefully  spread  over  the  surface  in  a  thin  film.     If  taken  from  a 
solid  medium,  a  small  drop  of  sterile  water  is  first  placed  upon  the 
cover-slip  and  the  bacteria  are  then  in  very  small  quantity  carefully 
emulsified  in  this  drop  with  the  platinum  needle  or  loop  and  spread 
in  an  extremely  thin  film. 

(2)  The  film  is  allowed  to  dry  in  the  air. 

(3)  When  thoroughly  dried,  fixation  is  carried  out  by  passing 
the  preparation,  film  side  up,  three  times  through  a  Bunsen  flame, 
at  about  the  rate  of  a  pendulum  swing.     Fixation  by  heat  in  this 
manner  is  most  convenient  for  routine  work,  but  is  not  the  most 
delicate  method,  inasmuch  as  the  degree  of  heat  applied  can  not  be 
accurately  controlled.     The  other  methods  which  may  be  employed 
are  immersion  in  methyl  alcohol,  formalin,  saturated  aqueous  bi- 
chloride of  mercury,  Zenker's  fluid,  or  acetic  acid.    If  chemical  fixa- 
tives are  used,  they  must  be  removed  by  washing  in  water  before 
the  stain  is  applied.    If  a  preparation  is  made  upon  a  slide  instead 
of  a  cover-slip,  passage  through  the  flame  should  be  repeated  eight 
or  nine  times. 

(4)  Staining. — The  dyes  used  for  the  staining  of  bacteria  are, 
for  the  greater  part,  basic  anilin  dyes,  such  as  methylene-blue,  gen- 
tian-violet, and  fuchsin.     These  may  be  applied  for  simple  staining 
in  5  per  cent  aqueous  solutions  made  up  from  filtered  saturated 
alcoholic  solutions,  or  directly  by  weight.     They  are  conveniently 
kept  in  the  laboratory  as  saturated  alcoholic  solutions.  The  strengths 
of  some  saturated  solutions  are  as  follows:3 

Fuchsin  (aqueous),  1.5  per  cent. 

Fuchsin  (alcohol  96  per  cent),  3  per  cent. 

Gentian- violet  (aqueous),  1.5  per  cent. 

Gentian-violet  (alcohol  96  per  cent),  4.8  per  cent. 

Methylene-blue  (aqueous),  6.7  per  cent. 

Methylene-blue  (alcohol  96  per  cent),  7  per  ,cent. 

The  staining  solution,  in  simple  routine  staining,  is  left  upon 
the  fixed  bacterial  film  for  from  one-half  to  one  and  one-half  minutes 
according  to  the  efficiency  of  the  stain  used.  Methylene-blue  is  the 
weakest  of  the  three  stains  mentioned ;  gentian- violet  the  strongest. 

(5)  The  excess  stain  is  removed  by  washing  with  water. 

3  After  Wood,  ' '  Chemical  and  Microscopical  Diagnosis, ' '  Appendix.  N.  Y.,  1909. 


114  BIOLOGY  AND  TECHNIQUE 

(6)  The  preparation  is  thoroughly  dried  by  a  blotter  or  between 
layers  of  absorbent  paper. 

(7)  A  small  drop  of  Canada  balsam  is  placed  upon  the  film  side 
of  the  dry  cover-slip,  which  is  then  inverted  upon  a  slide.     The 
preparation  is  now  ready  for  microscopical  examination. 

The  chemical  principles  which  underlie  the  staining  process  are 
still  more  or  less  in  doubt.4  Suffice  it  to  say  here  that  most  of  the 
dyes  in  common  use  by  bacteriologists  and  pathologists  are  coal-tar 
derivatives  belonging  to  the  aromatic  series,  all  of  them  containing 
at  least  one  "benzolring"  combined  with  what  Michaelis  terms  a 
' ' chromophore  group,"  chief  among  which  are  the  nitro-group 
(N02),  the  nitroso-group  (NO),  and  the  azo-group  (N=N).  Just 
what  the  actual  process  of  staining  consists  in  is  a  question  about 
which  various  opinions  are  held,  some  believing  that  the  phenomenon 
is  purely  chemical,  in  which  a  salt  is, formed  by  ihe  combination 
of  the  dye  and  the  protoplasm  of  the  cells,  others  that  there  is  no 
such  salt  formation,  and  that  the  process  takes  place  by  purely 
physical  means.  To  support  the  latter  view  it  is  argued  that  certain 
substances  like  cellulose  are  stainable  without  possessing  the  prop- 
erty of  salt  formation,  and  that  staining  may  often  be  accomplished 
without  there  being  a  chemical  disruption  of  the  dye  itself.  Michaelis 
sums  up  his  views  by  stating  that  probably  both  processes  actually 
take  place.  A  dye  stuff,  as  a  whole,  may  enter  into  and  be  deposited 
upon  a  tissue  or  cell  by  a  process  which  he  speaks  of  as  "insorp- 
tion. "  In  such  a  case  the  coloring  matter  may  be  subsequently 
extracted  by  any  chemically  indifferent  solvent,  On  the  other  hand, 
a  dye  after  being  thus  deposited  upon  or  within  a  cell,  may  become 
chemically  united  to  the  protoplasm  by  the  formation  of  a  salt, 
and  in  such  a  case  the  color  can  be  removed  only  by  agents  which 
are  capable  of  decomposing  salts,  such  as  free  acids. 

The  staining  power  of  any  solution  may  be  intensified  either  by 
heating  while  staining,  by  prolonging  the  staining  process,  or  by 
the  addition  of  alkalies,  acids,  anilin  oil,  and  other  substances  which 
will  be  mentioned  in  the  detailed  descriptions  of  special  staining 
methods. 


4  For  comprehensive  reviews  of  the  subject,  the  reader  is  referred  to  disserta- 
tions such  as  those  of  Mann  ("Physiol.  Hist.  Methods  and  Theory,"  Oxford, 
1902)  and  of  Michaelis  ("Einfuhrung  in  die  Farbstoffchemie, "  etc.,  Berlin, 
1902). 


MICROSCOPIC   STUDY  AND  STAINING  115 

In  addition  to  the  5  per  cent  aqueous  solutions  of  the  saturated 
alcoholic  solutions  of  the  dyes  mentioned  above,  a  few  extremely 
useful  stains  for  routine  work  are  as  follows : 

Loeffler's  A  Ikalin  Methylene-Blue : 

Saturated  alcoholic  methylene-blue 30  c.c. 

1  to  10,000  Potassium  hydrate  in  water 100  c.c. 

Carbol-Fuchsin':5 

Basic  fuchsin    1  gram 

Alcohol,  absolute    10     c.c. 

5%  Aqueous  carbolic  acid 90     c.c. 

To  make  up  this  stain  mix  90  c.e.  of  5  per  cent  aqueous  solution  of 
carbolic  acid  with  10  c.c.  saturated  alcoholic  basic  fuchsin. 
It  can  also  be  made  up  by  weighing  out : 

Basic  fuchsin   1    gram 

Carbolic  acid 5  grams 

Dissolve  in  distilled  water,  90  c.c.  filter  and  add  absolute  alcohol,  10  c.c. 
Toluidin-Blue  Solution. — A  very  useful  stain  for  general  bacteriological 
work  and  for  diphtheria  bacilli. 

Toluidin  blue   0.25  gram . 

Acetic    acid    2.0  c.c. 

Absolute  alcohol    , 5.0  c.c. 

Distilled  water   100.0  c.c. 

This  can  be  used  alone  when  it  gives  a  stain  like  Loeffler's  methylene  blue, 
but  more  clear,  or  else  a  counterstain  of  Bismarck  brown  can  be  used. 
Pappenheim-Saathof  Methylgreen: 

Methylgreen    0.15  grams 

Pyronin 0.5  grams 

95%    Alcohol    5.0       c.c. 

Glycerin  20.0       c.c. 

2%  Carbolic  acid  in  water  up  to 100.0       c.c. 

Stain  1  to  2  minutes,  wash,  blot.  This  is  a  splendid  method  of  staining 
bacteria  in  general  and  it  is  particularly  useful  for  the  staining  of  phagocytes 
containing  bacteria,  as  in  gonococcus  smears  and  in  opsonic  work. 

sSaathof,  Deut.  med.  woch.,  1905. 


116  BIOLOGY  AND  TECHNIQUE 

SPECIAL    STAINING   METHODS 

Spore  Stains. — ABBOTT'S  METHOD.6 — Cover-slips  are  smeared  and  fixed  by 
heat  in  the  usual  manner. 

Cover  with  Loeffler's  alkaline  methylene-blue  and  heat  the  stain  until  it 
boils,  repeat  the  heating  at  intervals  but  do  not  boil  continuously.  Keep 
this  up  for  one  minute. 

Rinse  in  water. 

Decolorize  with  a  mixture  of  alcohol  eighty  per  cent  98  c.c.  and  nitric 
acid  2  c.c.,  until  all  blue  has  disappeared. 

Rinse  in  water. 

Dip  from  three  to  five  seconds  in  saturated  alcoholic  solution  of  eosin 
10  c.c.,  and  water  90  c.c. 

Rinse  in  water,  blot,  and  mount  in  balsam. 

By  this  method  the  spores  are  stained  blue,  the  bodies  of  the  bacilli  are 
stained  pink. 

MOELLER'S  METHOD/ — Cover-slips  are  prepared  as  usual  and  fixed  in  the 
flame. 

Wash  in  choloroform  for  two  minutes. 

Wash  in  water. 

Coyer  with  five  per  cent  chromic  acid  one-half  to  two  minutes. 

Wash  in  water.  Invert  and  float  cover-slip  on  carbol-fuchsin  solution  in 
a  small  porcelain  dish  and  heat  gently  with  a  flame  until  it  steams;  continue 
this  for  three  to  five  minutes.  (This  step  can  also  be  done  by  covering  the 
cover-glass  with  carbol-fuchsin  and  holding  over  flame.) 

Decolorize  with  five  per  cent  sulphuric  acid  five  to  ten  seconds. 

Wash  in  water. 

Stain  with  aqueous  methylene-blue  one-half  to  one  minute.  By  this 
method  spores  will  be  stained  red,  the  body  blue. 

Capsule  Stains. — WELCH'S  METHOD.S — Cover-slips  are  prepared  as  usual 
but  dried  without  heat. 

Cover  with  glacial  acetic  acid  for  a  few  seconds.     Pour  off  acetic  acid 
and  cover  with  anilin  water  gentian-violet,  renewing  stain  repeatedly  until 
all  acid  is  removed.     This  is  done  by  pouring  the  stain  on  and  off  three  or 
four  times  and  then  finally  leaving  it  on  for  about  three  minutes. 
Wash  in  two  per  cent  salt  solution  and  examine  in  this  solution. 
Hiss'  METHODS.9 — (1)  Copper  Sulphate  Method. — Cover-slip  preparations 


e  Abbott,  "Prin.  of  Bact.,"  Phila.,  1905. 

7  Moeller,  Cent,  f .  Bakt.,  I,  x,  1891. 

8  Welch,  Johns  Hopkins  Hosp.  Bull.,  1892,  iii,  81. 

8  Hiss.  Cent.  f.  Bakt.,  xxxi,  1902;  Jour.  Exper.  Mod.,  vi,  1905. 


MICROSCOPIC  STUDY  AND  STAINING  117 

are  made  by  smearing  the  organisms  in  a  drop  of  animal  serum,  preferably 
beef-blood  serum. 

Dry  in  air  and  fix  by  heat. 

Stain  for  a  few  seconds  with — 

Saturated  alcoholic  solution  of  fuchsin  or  gentian-violet  5  c.c.,  in  distilled 
water  95  c.c. 

The  cover-slip  is  flooded  with  the  dye  and  the  preparation  held  for  a  second 
over  a  free  flame  until  it  steams. 

Wash  off  dye  with  twenty  per  cent  aqueous  copper  sulphate  solution. 

Blot  (do  not  wash). 

Dry  and  mount. 

By  this  method  permanent  preparations  are  obtained,  the  capsule  appear- 
ing as  a  faint  blue  halo  around  a  dark  purple  cell  body. 

HUNTOON'S  CAPSULE  STAIN  (applicable  only  to  cultures,  not  to  animal 
exudates). — This  depends  upon  the  precipitating  action  of  lactic  acid  on 
nutrose.  Requires  two  solutions. 

1.  Diluent. — 3  per  cent  solution  of  nutrose  in  distilled  water;  place  in  Arnold 
one  hour,  add  a  small  amount  of  carbolic  as  preservative,  and  allow  to  settle. 

2.  Stain  and  fixative. — 2  per  cent  carbolic,  100  c.c.;   concentrated  lactic  acid, 
0.5  x;.c. ;  1  per  cent  acetic  acid,  1  c.c. ;  saturated  alcoholic  solution  basic  fuchsin, 
1  c.c.;  carbol  fuchsin,  1  c.c. 

As  to  the  dye  employed,  most  anything  but  methylene  blue  or  Bismarck  brown 
may  be  used.  Methyl  violet  gives  the  most  beautiful  results  but  is  not  permanent 
and  will  not  photograph.  I  have  found  the  above  mixture  the  best  for  classroom 
work. 

Make  a  thin  film,  employing  solution  1  as  diluent.  Dry  in  air.  Do  not 
fix.  Cover  with  stain  30  seconds.  Wash  in  water,  dry,  and  examine. 

BUERGER'S  METHOD.™ — Cover-slip  preparations  are  made  by  smearing  in 
serum  as  in  Hiss'  method. 

As  the  edges  of  the  smear  begin  to  dry,  pour  over  it  Zenker's  fluid  (with- 
out acetic  acid)  and  warm  in  flame  for  three  seconds. 

(Zenkers  fluid  is  composed  of  potassium  bichromate  2.5  gm.,  sodium 
sulphate  1  gm.,  water  100  c.c.,  saturated  with  bichloride  of  mercury.) 

Wash  in  water. 

Flush  with  ninety-five  per  cent  alcohol. 

Cover  with  tincture  of  iodin,  U.  S.  P.,  one  to  three  minutes. 

Wash  with  ninety-five  per  cent  alcohol. 

Dry  in  the  air. 

Stain  with  anilin  water  gentian-violet  two  to  five  seconds. 

Wash  with  two  per  cent  salt  solution. 

Mount  and  examine  in  salt  solution. 


Buerger,  Med.  News,  Doc.,  1904. 


118  BIOLOGY  AND  TECHNIQUE  . 

WADSWORTH'S  METHOD.11 — Wadsworth  has  devised  a  method  of  staining 
capsules  which  depends  upon  the  fixation  of  smears  with  formalin.  After 
such  fixation  capsules  may  be  demonstrated  both  with  simple  stains  and  by 
Gram's  method.  The  technique  is  as  follows: 

Smear  preparations,  made  as  usual,  are  treated  as  follows: 

1.  Formalin,  40  per  cent,  two  to  five  minutes. 

2.  Wash  in  water,  five  seconds. 

SIMPLE  STAIN.  DIFFERENTIAL  STAIN  (Gram's  Method). 

3.  Ten  per  cent  aqueuos  gentian-violet.       3.  Anilin  gentian-violet,  two  minutes. 

4.  Wash  water,  five  seconds.  4.  lodin  'solution,  two  minutes. 

5.  Dry,  mount  in  balsam.  5.  Alcohol,  95  per  cent,  decolorize. 

6.  Fuchsin,  dilute  aqueous  solution. 

7.  Wash  water,  two  seconds. 

8.  Dry,  mount  in  balsam. 

It  is  important  that  the  formalin  be  fresh  and  the  exposure  to  water 
momentary.  When  decolorizing  in  the  Gram  method,  strong  alcohol  only 
should  be  used.  Wadsworth  also  found  that  encapsulated  pneumococci  could 
be  demonstrated  in  celloidin  sections  of  pneumonic  lesions  hardened  in  strong 
formalin.  The  lungs  should  be  distended  with  the  formalin  or  the  lesions 
cut  in  very  thin  bits,  hardened,  dehydrated,  embedded,  and  cut  in  the  usual 
way.  The  celloidin  sections  may  be  fixed  on  the  slides  by  partially  dis- 
solving the  celloidin  in  alcohol  and  ether  and  setting  the  celloidin  quickly 
in  water  before  staining.  Failure  to  obtain  pneumococci  encapsulated  in 
such  sections  is  usually  due  to  improper  or  inadequate  fixation  in  the  formalin. 

The  differential  method  employed  by  Wadsworth  for  tissue  staining  is 
as  follows: 

1.  Fix  in  formalin  forty  per  cent,  two  to  five  minutes. 

2.  Wash  in  water. 

3.  Anilin  gentian-violet,  two  minutes. 

4.  lodin  solution,  two  minutes. 

5.  Alcohol,  ninety-five  per  cent,  decolorize. 
•6.  Eosin  alcohol,  counterstain. 

7.  Clear  in  oil  of  origanum. 

8.  Mount  in  balsam. 

Flagella  Stains.— All  flagella  stains,  in  order  to  be  successful,  necessitate 
particularly  clean  cover-slip  preparations,  best  made  from  young  agar  cul- 
tures emulsified  in  sterile  salt  solution.  Scrupulous  care  should  be  exercised 
in  cleaning  the  glassware  used. 


11  Wadsworth,  Jour.  Inf.  Dis.,  1906. 


MICROSCOPIC   STUDY   AND   STAINING  119 

LOEFFLER'S  METHOD.12 — The  preparation  is  dried  in  the  air  and  fixed  by 
heat.  It  is  then  treated  with  the  following'  mordant  solution: 

Twenty  per  cent  aqueous  tannic  acid 10  parts. 

Ferrous  sulphate  aq.  sol.  saturated  at  room  temperature. .  .     5  parts. 
Saturated  alcoholic  fuchsin  solution 1  part. 

This  solution  which  should  be  freshly  filtered  before  using,  is  poured  over 
the  cover-glass  and  allowed  to  remain  there  for  one-half  to  one  minute, 
during  which  time  it  should  be  gently  heated,  but  not  allowed  to  boil. 

Wash  thoroughly  in  water. 

Stain  with  five  per  cent  anilin  water  fuchsin  or  anilin  water  gentian-violet 
made  slightly  alkaline  by  the  addition  of  one-tenth  per  cent  sodium  hydrate. 

The  stain  should  be  filtered  directly  upon  the  cover-slip.  Warm  gently 
and  leave  on  for  one  to  two  minutes.  Wash  in  water.  Mount  in  balsam. 

VAN  ERMENGEM'S  METHOD.IS — This  method  requires  the  preparation  of 
three  solutions. 

(1)  Twenty  per  cent  tannic  acid  solution 60  c.c. 

Two  per  cent  osmic  acid  solution 30  c.c. 

Glacial  acetic  acid 4-5  drops 

The  cover-slip  carrying  the  fixed  preparation  is  placed  in  this  solution 
for  one  hour  at  room  temperature,  or  for  five  minutes  at  100°  C.  (boiling). 
Wash  in  water. 
Wash  in  absolute  alcohol. 
Immerse  the  cover-slip  for  one  to  three  seconds  in 

(2)  Silver  nitrate,  0.25-0.5  per  cent  solution. 
Without  washing,  transfer  to 

(3)  Gallic  acid 5  gin. 

Tannic  acid 3    " 

Fused   potassium   acetate 10    lt 

Distilled  water 350  c.c. 

Immerse  in  this  for  a  few  minutes,  moving  the  cover-slip  about. 

Return  to  the  silver  nitrate  solution  until  the  preparation  turns  black. 

Wash  thoroughly  in  water. 

Blot  and  mount. 

SMITH'S  MODIFICATION  OF  PITFIELD'S  METHOD.14 — A  saturated  solution 
of  bichlorid  of  mercury  is  boiled  and  is  poured  while  still  hot  into  a  bottle 
in  which  crystals  of  ammonia  alum  have  been  placed  in  quantity  more  than 


12  Loeffler,  Cent,  f .  Bakt.,  I,  vi,  1889. 

18  Van  Ermengem,  Cent.  f.  Bakt.,  I,  xv,  1894. 

14  Smith,  Brit.  Med.  Jour.,  I,  1901r  p.  205. 


120  BIOLOGY  AND  TECHNIQUE 

sufficient  to  saturate  the  fluid.  The  bottle  is  then  shaken  and  allowed  to 
cool.  Ten  c.c.  of  this  solution  are  added  to  10  c.c.  of  freshly  prepared  ten 
per  cent  tannic  acid  solution.  To  this  add  5  c.c.  carbol-fuchsin  solution. 
Mix  and  filter. 

To  stain,  filter  the  above  mordant  directly  upon  the  fixed  cover-slip 
preparation.  Heat  gently  for  three  minutes,  but  do  not  allow  to  boil.  Wash 
in  water  and  stain  with  the  following  solution : 

Saturated  alcoholic  solution  gentian >violet 1  c.c. 

Saturated  solution  ammonia  alum 10  c.c. 

Filter  the  stain  directly  upon  the  preparation  and  heat  for  three  or  four 
minutes.  Wash  in  water,  dry,  and  mount  in  balsam. 

Differential  Stains. — GRAM'S  METHOD.IS — By  this  method  of 
staining,  which  is  extremely  important  in  bacterial  differentiation, 
bacteria  are  divided  into  those  which  retain  the  initial  stain  and 
those  which  are  subsequently  decolorized  and  take  the  counterstain. 
The  former  are  often  spoken  of  as  the  Gram-positive,  the  latter  as 
Gram-negative  bacteria. 

The  reasons  for  the  differential  value  of  Gram's  method  are  not 
entirely  clear,  but  must,  of  course,  depend  upon  peculiarities  of  the 
chemical  constituents  of  the  bacteria  themselves.  A  considerable 
amount  of  work  has  been  done  on  the  subject  which  has  not  been 
entirely  conclusive.  A  discussion  of  the  subject  may  be  found  in 
Wells'  "Chemical  Pathology,"  Second  Edition,  page  105.  Wells 
states  that  the  results  of  Gram's  method  may  be  ascribed  to  the 
formation  of  an  iodin-pararosanilin-protein  compound  in  the  Gram- 
positive  bacteria.  Only  dyes  of  this  group,  namely,  gentian  violet, 
methyl  violet,  etc.,  will  form  such  combination.  He  quotes  Burgers16 
as  stating  that  trypsin  will  digest  Gram-negative,  but  not  Gram- 
positive  bacteria,  and  the  gastric  juice  affects  only  a  few  of  the 
Gram-positive  species.  It  has  also  been  suggested  that  the  fatty 
substances  in  the  bacterial  bodies  may  have  some  relationship  to 
the  Gram-stain,  and  that  the  bacterial  cell  wall  is  the  part  most 
directly  involved  in  the  staining  reaction.  Benians17  has  found  that 
when  Gram-positive  bacteria  are  artificially  disintegrated  they  lose 
their  characteristic  reaction  and  become  Gram-negative,  and  this 
has  been  confirmed  for  tubercle  bacilli  in  Wells'  laboratory  by 

15  Gram,  Fortschr.  d.  Med.,  ii,  1884. 

18  Burgers  and  collaborators,  Zeit.  f.  Hyg.,  1911,  70'. 

17  Benians,  Jour,  of  Pathol.  and  Bacter.,  17,  1912,  199. 


MICROSCOPIC   STUDY  AND   STAINING  121 

Sherman.18  Wells'  suggestion  is  that  the  iodin  may  render  the  cell 
membrane  impermeable  to  alcohol. 

Preparations  are  made  on  cover-slips  or  slides  in  the  usual  way. 

The  preparation  is  then  covered  with  an  anilin  gentian-violet 
solution  which  is  best  made  up  freshly  before  use. 

The  staining  fluid  is  made  up,  according  to  Gram's  original  direc- 
tions,19 as  follows: 

Five  c.c.  of  anilin  oil  are  shaken  up  thoroughly  with  125  c.c.  of  distilled 
water.  This  solution  is  then  filtered  through  a  moist  filter  paper. 

To  108  c.c.  of  this  anilin  water,  add  12  c.c.  of  a  saturated  alcoholic 
solution  of  gentian-violet.  The  stain  acts  best  when  twelve  to  twenty-four 
hours  old,  but  may  be  used  at  once.  It  lasts,  if  well  stoppered,  for  three 
to  five  days.  A  more  convenient  and  simple  method  of  making  up  the  stain 
is  as  follows: 

To  10  c.c.  of  distilled  water  in  a  test  tube  add  anilin  oil  until  on  shaking 
the  emulsion  is  opaque ;  roughly,  one  to  ten.  Filter  this  through  a  wet  paper 
until  the  filtrate  is  clear.  To  this  add  saturated  alcoholic  solution  of  gentian- 
violet  until  the  mixture  is  no  longer  transparent,  and  a  metallic  film  on 
the  surface  indicates  saturation.  One  part  of  alcoholic  saturated  gentian- 
violet  to  nine  parts  of  the  anilin  water  will  give  this  result.  This  mixture 
may  be  used  immediately  and  lasts  two  to  five  days  if  kept  in  a  stoppered 
bottle. 

Cover  the  preparation  with  this;  leave  on  for  5  minutes.  Pour  off  excess 
stain  and  cover  with  Gram's  iodin  solution  for  2  to  3  minutes. 

Iodin 1  gm. 

Potassium  iodid 2  gm. 

Distilled  water 300  c.c. 

Decolorize  with  ninety-seven  per  cent  alcohol  until  no  further  traces  of 
the  stain  can  be  washed  out  of  the  preparation.  This  takes  usually  thirty 
seconds  to  two  minutes,  according  to  thinness  of  preparation. 

Wash  in  water. 

Counterstain  with  an  aqueous  contrast  stain,  preferably  Bismarck  brown,20 
dilute  fuchsin  or  safranin. 

PALTAUF'S  MODIFICATION.  OF  GRAM'S  STAIN. 21 — The  staining  fluid 
as  prepared  by  this  modification  possesses  the  advantage  of  retaining 


18  Sherman,  Jour,  of  Infec.  Dis.,  12,  1913,  249. 

19  Gram,  loc.  cit. 

20  To  make  up  Bismarck  brown  solution,  prepare  a  saturated  aqueous  solution 
of  the  powdered  dye  by  heating.     Cool  and  filter.     Dilute  1  to  10  with  distilled 
water. 

21Sharnosky,  Proc.  N.  Y.  Pathol.  Soc.,  Oct.,  1909,  n.  s.,  ix,  5, 


122  BIOLOGY  AND  TECHNIQUE 

its  staining  power  for  a  longer  period  than  does  the  anilin-water 
gentian-violet  described  in  the  original  method. 

The  staining  fluid  is  prepared  as  follows: 

3-5  c.c.  anilin  oil  are  added  to 

90  c.c,  distilled  water  and 

7  c.c.  absolute  alcohol. 

This  mixture  is  thoroughly  shaken  and  filtered  through  a  moist  filter 
paper  until  clear.  Then  add: 

Gruebler's  gentian-violet  2  gm. 

The  fluid  should  stand  twenty-four  hours,  during  which  a  precipitate 
forms.  This  is  filtered  before  use. 

This  gentian-violet  solution  retains  its  staining  power  for  from  4  to  6 
weeks.  It  is  good  only  when  a  metallic  luster  develops  on  the  surface. 

It  is  used  in  the  following  way:  Spreads  on  cover-slips  or  slides  are 
dried  and  fixed  as  usual. 

Then  apply: 

Anilin-water  gentian-violet  (as  above),  three  minutes'. 

Gram's  iodin  solution,  two  minutes. 

Absolute  alcohol  (with  stirring),  thirty  seconds. 

Counterstain,  without  washing  in  water,  in  aqueous  fuchsin  or  in  weak 
carbol-fuchsin. 

Jensen's  Modification  of  the  Gram  Stain*2 — Jensen  prepares  his  smears 
in  the  ordinary  way.  He  uses  a  5  per  cent  solution  of  methyl  violet,  "6B." 
This  solution  is  stable.  It  is  poured  on  the  fixed  smear  for  *4  to  l/2  minute. 
The  methyl  violet  solution  is  poured  off  and,  without  washing,  is  covered 
with  LugoPs  solution,  and  this  is  poured  off  and  fresh  Lugol  added  and  left 
on  for  1/2  minute. 

The  iodine  solution  is  now  poured  off  and  the  preparation  washed  with 
98%  alcohol.  When  decolorized,  the  98%  alcohol  is  washed  off  with  a  few 
drops  of  absolute  alcohol,  and  the  preparation  counterstained  with  a  solution 
of  neutral  red  consisting  of  1  gram  of  neutral  red  in  a  liter  of  water  to 
which  2  c.c.  glacial  acetic  acid  has  been  added. 

Apparently  the  important  point  in  Jensen's  stain  is  that  the  preparation 
is  never  washed  with  water,  and  that  the  final  washing  off  of  the  98°  alcohol 
is  done  with  a  few  drops  of  absolute. 

We  have  not  used  this  stain  as  a  routine  and  do  not  know  how  reliable 
it  is. 

STERLING'S  MODIFICATION  OF  0 RAM'S  METHOD. — 2  c.c.  anilin  oil  + 
10  c.c.  95%  alcohol.  Shake  ami  add  88  c.c.  distilled  water.  5  grams 


23  Jensen,  Berl.  klin.  Woch.,  49,  1912,  1163. 


MICROSCOPIC  STUDY   AND   STAINING 


123 


gentian  violet  are  ground  in  a  mortar  and  the  anilin  solution  added 
slowly  while  grinding.  Filter.  This  solution  keeps,  and  stains  in 
one-half  to  one  minute.23 

CLASSIFICATION    OF    THE    MORE    IMPORTANT    PATHOGENIC    BACTERIA 
ACCORDING  TO  GRAM'S  STAIN. 


Gram-positive. 
(Retain  the  Gentian-violet.) 

Micrococcus  pyogenes  aureus 

Micrococcus  pyogenes  albus 

Streptococcus  pyogenes 

Micrococcus  tetragenus 

Pneumococcus 

Bacillus  subtilis 

Bacillus  anthracis 

Bacillus  diphtherias 

Bacillus  tetanus 

Bacillus  tuberculosis  and  other 

acid-fast  bacilli 
Bacillus  aerogenes  capsulatus 
Bacillus  botulinus 


Gram-negative. 
(Take  Counter  stain.) 

Meningococcus 
Gonococcus 

Micrococcus  catarrhalis 
Bacillus  coli 
Bacillus  dysenteriae 
Bacillus  typhosus 
Bacillus  paratyphosus 
Bacillus  fecalis  alkaligenes 
Bacillus  enteritidis 
Bacillus  proteus   (proteus) 
Bacillus  mallei 
Bacillus  pyocyaneus 
Bacillus  influenzas 
Bacillus  mucosus  capsulatus 
Bacillus  pestis 
Bacillus  maligni  oedematis 
Spirillum  choleras 
Bacillus  Koch- Weeks 
Bacillus  Morax-Axenfeld 


Stains  for  Acid-Fast  Bacteria. — These  methods  of  staining  are 
chiefly  useful  in  the  demonstration  of  tubercle  bacilli.  These  bacteria 
because  of  their  waxy  cell  membranes  are  not  easily  stained  by  any 
but  the  most  intensified  dyes,  but  when  once  stained,  retain  the 
color  in  spite  of  energetic  decolorization  with  acid.  For  this  reason 
they  are  known  as  acid-fast  bacilli.  The  first  method  devised  for 
the  staining  of  tubercle  and  allied  bacilli  was  that  of  Ehrlich. 

23  This  is  the  routine  method  employed  in  our  laboratory  at  present.  In  using 
Sterling's  stain  the  time  of  staining  can  be  abbreviated  as  follows: 

Sterling's  Gentian  Violet one  minute 

lodin    thirty  seconds  to  one  minute 


124  BIOLOGY  AND  TECHNIQUE 

EHRLICH  METHOD.24 — This  method  is  now  rarely  used.  Cover-slip 
preparations  are  prepared  as  usual  and  fixed  by  heat. 

Stain  with  anilin  water  gentian-violet,  hot,  three  to  five  minutes,  or 
twenty-four  hours  at  room  temperature. 

Decolorize  with  thirty-three  per  cent  nitric  acid  one-half  to  one  minute. 
Treat  with  sixty  per  cent  alcohol,  until  no  color  can  be  seen  to  come  off. 
Counterstain  with  aqueous  methylene-blue. 
Rinse  in  water,  dry,  and  mount. 

ZIEHL-NEELSON  METHOD.25 — Thin  smears  are  made  upon  cover- 
slips  or  slides. 

Fix  by  heat. 

Stain  in  carbol-fuchsin  solution  as  given  on  page  115.  The  slide  or  cover- 
slip  may  be  flooded  with  the  stain,  and  this  gently  heated  with  the  flame 
until  it  steams,  or  else  the  cover-slip  may  be  inverted  upon  the  surface  of 
the  staining  fluid,  in  a  porcelain  dish  or  watch-glass,  and  this  heated  until 
it  steams.  This  is  continued  for  three  to  five  minutes.  Decolorize  with 
either  five  per  cent  nitric  acid,  five  per  cent  sulphuric  acid,  or  one  per  cent 
hydrochloric  acid  for  three  to  five  seconds.  The  treatment  with  the  acid  is 
continued  until  subsequent  washing  with  water  will  give  only  a  faint  pink 
color  to  the  preparation. 

Wash  with  ninety  per  cent  alcohol  until  no  further  color  can  be  removed. 
If,  after  prolonged  washing  with  alcohol,  a  red  color  still  remains  in  very 
thick  places  upon  the  smear,  while  the  thin  areas  appear  entirely  decolorized, 
this  may  be  disregarded. 

Wash  in  water  and  counterstain  in  aqueous  methylene-blue  for  one-half 
to  one  minute. 

Rinse  in  water,  dry,  and  mount. 

By  this  method  the  tubercle  bacilli  are  colored  red,  other  bacteria  and 
cellular  elements  which  may  be  present  are  stained  blue. 

GABBET'S  METHOD. 26— Gabbet  has  devised  a  rapid  method  in 
which  the  decolorization  and  counterstaming  are  accomplished  by 
one  solution.  The  specimen  is  prepared  and  stained  with  carbol- 
fuchsin  as  in  the  preceding  method.  It  is  then  immersed  for  one 
minute  directly  in  the  following  solution : 

Methylene-blue    2  gnis. 

Sulphuric  acid  25  per  cent  (sp.  gr.  1018) 100    c.c. 

MEhrlich,  Deut.  med.  Woch.,  1882. 

28  Ziehl,  Deut.  med.  Woch.,  1882  •  Neelson,  Deut.  med.  Woch.,  1883. 

26  Goblet,  Lancet,  1887. 


MICROSCOPIC   STUDY  AND  STAINING  125 

Then  rinse  in  water,  dry,  and  mount. 

This  method,  while  rapid  and  very  convenient,  is  not  so  reliable  as  the 
Ziehl-Neelson  method. 

PAPPENHEIM  's  METHOD.27 — The  method  of  Pappenheim  is  devised 
for  the  purpose  of  differentiating  between  the  tubercle  bacillus  and 
the  smegma  bacillus.  Confusion  may  occasionally  arise  between 
these  two  microorganisms,  especially  in  the  examination  of  urine 
where  smegma  bacilli  arc  derived  from  the  genitals,  and  less  fre- 
quently in  the  examination  of  sputum  where  smegma  bacilli  may 
occasionally  be  mixed  with  the  secretions  of  the  pharynx  and  throat. 

Preparations  are  smeared  and  fixed  by  heat  in  the  usual  way. 

Stain  with  hot  carbol-fuchsin  solution  for  two  minutes. 

Pour  off  dye  without  washing  and  cover  with  the  following  mixture: 

Corallin    (rosolic  acid)    1  gm. 

Absolute    alcohol    100    c.c. 

Methylene-blue  added  to  saturation 

Add  glycerin28  20    c.c. 

This  mixture  is  poured  on  and  drained  off  slowly,  the  procedure  being 
repeated  four  or  five  times,  and  finally  the  preparation  is  washed  in  water. 
The  combination  of  alcohol  and  rosolic  acid  decolorizes  the  smegma  bacilli, 
but  leaves  the  tubercle  bacilli  stained  bright  red. 

BAUMGARTEN'S  METHOD.29 — This  method  is  recommended  by  the 
author  for  differentiation  between  the  bacillus  of  tuberculosis  and 
the  bacillus  of  leprosy  and  depends  upon  the  fact  that  the  tubercle 
bacillus  is  less  easily  stained  than  Bacillus  leprse. 

Smears  are  prepared  and  fixed  by  heat  in  the  usual  way. 

Stain  in  dilute  alcoholic  fuchsin  for  five  minutes. 

Decolorize  for  twenty  seconds  in  alcohol,  ninety-five  per  cent,  ten  parts, 
nitric  acid  one  part. 
Wash  in  water. 

Counterstain  in  methylene-blue. 
Wash  in  water,  dry,  and  mount. 


27  Pappenheim,  Bcrl.  klin.  Woch.,  1898. 

28  The  glycerin  is  added  after  the  other  constituents  have  been  mixe<L 
™Baumgarten,  Zeit.  f.  wissensch.  Mikrosk.,  1,  1884. 


126  BIOLOGY  AND  TECHNIQUE 

The  tubercle  bacillus  should  be  blue  and  the  bacillus  of  leprosy  red. 
Hermann's  Stain  for  Acid  Fast  Bacteria:  30 

I.  Crystal  Violet  3%  in  alcohol. 
II.  Ammonium  Carbonate  1%  aqueous. 

Mix  one  part  of  "I"  with  3  parts  of  "II"  just  before  using.  Steam 
3  minutes.  Decolorize  with  10%  nitric  acid  (or  5%  sulphuric  acid).  Wash 
in  alcohol,  rinse  and  counterstain  with  Bismarck  brown.  The  better  contrast 
obtained  with  this  stain  is  stated  by  Park  to  increase  the  ease  with  which 
tubercle  bacilli  can  be  found  in  sputum. 

Special  Stains  for  Polar  Bodies. — These  staining  methods  are 
designed  to  bring  into  view  polar  bodies  as  found,  for  instance,  in 
the  bacilli  of  diphtheria  and  plague. 

NEISSER'S  METHOD.31 — Smear  and  fix  in  the  usual  manner. 

Stain  for  two  to  five  seconds  in  the  following  solution : 

Methylene-blue   1  gm. 

Absolute   alcohol    1 20  c.c. 

Glacial   acetic  acid 50  c.c. 

Distilled  water   1,000  c.c. 

Wash  in  water. 

Counterstain  in  0.4  per  cent  aqueous  Bismarck  brown  solution 
for  five  seconds. 

By  this  method  polar  bodies  are  stained  blue,  while  the  bacillary 
bodies  are  stained  brown. 

Carbol  Thionin: 

Saturated  solution  of  thionin  in  50%  alcohol 10  c.c. 

2%  aqueous  solutions  of  phenol 100  c.c. 

Stain  one  to  three  minutes. 

See  also  Toluidin  Blue  solution  given  on  page  115. 

Polychrome  Stains. — The  various  polychrome  stains  are  of  value 
to  the  bacteriologist  chiefly  for  the  staining  of  pus  and  exudates 
where  the  relation  of  bacteria  to  cellular  elements  is  to  be  demon- 
strated. They  are  also  extremely  useful  in  the  study  of  fixed  speci- 
mens of  protozoan  parasites.  There  is  a  large  number  of  these 
stains  in  use ;  a  few  only,  however,  can  be  given  here.  In  principle, 


80  Quoted    from    Park    and    Williams,    Pathogenic    Microorganisms,    Lea    and 
Fcbiger,  Phila.,  1920. 

31  Neisser,  Zeit,  f,  Hyg.,  xxiv,  1897. 


MICROSCOPIC  STUDY  AND  STAINIXC,  12? 

all  these,  stains  depend  upon  a  combination  of  eosin  and  methylene- 
blue,  these  elements  staining  not  only  as  units,  but  acting  together 
in  combination.  One  and  the  same  solution,  therefore,  contains  a* 
least  three  elements  which  color  the  various  structures  of  the 
preparation  selectively. 

JENNER'S  METHOD.32 — This  stain,  because  of  its  simplicity,  is  use- 
ful for  routine  use.  It  is  made  up  as  follows :  Equal  parts  of  eosin 
(Gruebler,  "W.  G. ")  one  and  two-tenths  per  cent  aqueous  solution, 
and  methylene-blue  (medicinal,  Gruebler)  one  per  cent  aqueous 
solution,  are  mixed  and  allowed  to  stand  for  twenty-four  hours. 
A  coarse  granular  precipitate  is  formed  which  appears  dark,  with 
a  metallic  luster  on  its  surface.  This  is  separated  by  filtration  and 
washed  with  distilled  water  until  the  filtrate  appears  almost  clear. 

To  make  up  the  stain  0.5  gram  of  the  dry  precipitate  is  dissolved 
in  100  c.c.  of  methyl  alcohol. 

In  using  the  stain,  preparations  are  not  fixed,  but  simply  dried 
in  the  air  and  immersed  in  the  stain  for  one  to  two  minutes.  After 
this,  wash  in  distilled  water  and  examine. 

WRIGHT'S  MODIFICATION  OF  LEISHMAN'S  METHOD.33 — A  one  per 
cent  solution  of  methylene-blue  (Gruebler)  in  five-tenths  per  cent 
solution  of  sodium  bicarbonate  in  distilled  water  is  steamed  in  a 
sterilizer  at  100°  C.  for  one  hour.  After  this  has  cooled,  a  one-tenth 
per  cent  aqueous  solution  of  eosin  (Gruebler,  W.  G.)  is  added  until 
a  metallic  scum  appears  on  the  surface  of  the  mixture.  (About  five 
parts  of  eosin  solution  to  one  of  methylene-blue  is  necessary.)  The 
precipitate  which  forms  is  collected  by  filtration,  dried,  and  a 
saturated  solution  then  made  in  methyl  alcohol.  This  is  filtered 
and  diluted  with  one-quarter  its  bulk  of  methyl  alcohol. 

To  stain,  cover  the  dried  preparation  with  the  stain  for  one  to 
one  and  one-half  minutes.  Dilute  by  dropping  upon  the  stain  dis- 
tilled water  from  a  pipette  until  a  metallic  film  appears  upon  the 
top.  Leave  this  on  for  three  to  fifteen  minutes.  Wash  in  distilled 
water. 

GIEMSA'S  METHOD.S* — The  method  of  Giemsa  is  really  a  modifica- 
tion of  the  Komanowsky  method.  It  is  widely  applicable,  being  of 
great  value  in  the  staining  of  the  Spirochaste  pallida,  Vincent's 


32  Jenner,  Lancet,  i,  1889. 

33  Wright,  Jour.  Med.  Kesearch,  ii,  1902. 

,  Cent.  f.  Bakt.,  I,  xxxvii,  1904. 


128  BIOLCKJY  AND  TECHNIQUE 

spirilla,  protozoa,  and  Negri  bodies.  The  stain  has  been  modified 
several  times  by  its  originator,  the  following  being  the  formula,  given 
by  him  in  1904:  The  substance  referred  to  as  azur  II  and  purchas- 
able under  that  name,  consists  of  pure  methylenazur  chloralhydrate 
combined  with  an  equal  quantity  of  methylene-blue  chloralhydrate. 
The  substance  referred  to  as  azur  II-eosin  is  a  combination  of  this 
substance  with  eosin. 

The  staining  fluid  is  made  up  as  follows  :35 

Azur  II-eosin    3  gins. 

Azur  II 3  gms. 

This  mixture  is  thoroughly  dried  over  sulphuric  acid  in  a  desic- 
cator, finely  powdered,  and  rubbed  through  a  fine  sieve.  It  is  then 
dissolved  in  250  gms.  of  C.  P.  glycerin  (Merck),  at  60°  C.  To  this 
is  added  methyl  alcohol  (Kahlbaum)  250  c.c.,  previously  warmed 
to  60°  C.  This  mixture  is  well  shaken  and  allowed  to  stand  at  room 
temperature  for  twenty-four  hours.  The  mixture  is  now  ready  for 
use. 

For  use  10  c.c.  of  distilled  water  are  poured  into  a  test  tube  and 
one  to  two  drops  of  a  one  per  cent  potassium  carbonate  solution 
are  added.  Ten  drops  of  the  staining  solution  described  above  (one 
drop  to  the  c.c.)  are  mixed  with  this  slightly  alkaline  water.  The 
preparation  which  is  to  be  stained  is  fixed  in  methyl  alcohol,  dried, 
and  covered  with  the  diluted  staining  solution.  For  the  staining 
of  protozoa  and  exudates  containing  bacteria,  ten  to  fifteen  minutes 
are  sufficient.  For  the  staining  of  Negri  bodies  or  Spirochgete  pallida, 
one  or  more  hours  of  staining  should -be  employed.  After  staining, 
wash  in  running  tap  water  and  blot. 

WOOD'S  METHOD.36 — "Wood  has  devised  a  simple  staining  method 
based  on  the  principles  of  the  Giemsa  stain,  in  which  azur  II  and 
eosin  may  be  used  in  separate  solutions.  Preparations  are  fixed  in 
strong  methyl  alcohol  for  five  minutes  and  are  then  stained  in  a 
0.1  per  cent  aqueous  solution  of  eosin  until  the  preparation  is  pink. 
The  eosin  is  then  poured  off  and  the  preparation  is  covered  with  a 
0.25  per  cent  aqueous  solution  of  azur  II  for  one-half  to  two  minutes. 
Following  this,  it  is  washed  in  tap  water  and  dried  by  blotting. 

35  It  is  best  not  to  attempt  to  make  up  the  undiluted  staining  fluid,  since  this 
is  purchasable  under  the  nume  of  "Giomsa  Losnng  fiir  Romanowsky  Farbung. " 
38  Wood,  Mod.  News,  83,  1903. 


MICROSCOPIC  STUDY  AND  STAINING  129 

When  an  intense  stain  is  desired,  the  solution  of  eosin  and  azur 
II  may  be  flooded  over  the  preparation  together,  using  an  excess 
of  azur  II.  They  are  then  left  on  from  five  to  ten  minutes.  At  the 
end  of  this  time  washing  and  drying  as  before  complete  the  process. 

The  Staining*  of  Bacteria  in  Tissues. — The  preparation  of  tissue 
for  bacterial  staining  is,  in  general,  the  same  as  that  employed  for 
purposes  of  cellular  studies,  in  histology.  For  bacteriological  studies 
the  most  useful  are  alcohol  and  Zenker's  fluid.  Other  fixations, 
such  as  that  by  formalin,  or  Mueller's  fluid,  give  less  satisfaction. 
In  other  respects  the  details  of  dehydration  and  embedding  are  the 
same  as  those  used  in  histological  studies,  except  that  it  is  desirable 
that  the  tissues  should  be  handled  rather  more  carefully  than  is 
necessary  for  ordinary  pathological  work,  and  the  changes  from 
the  weaker  to  the  stronger  alcohols  should  be  made  less  abruptly.37 

Embedding  in  paraffin  is  preferable  to  celloidin,  although  the 
latter  method  is  not  unsuccessful  if  carefully  carried  out.  The  chief 
disadvantages  of  celloidin  are  the  retention  of  color  by  the  celloidin 
itself  and  the  consequent  unclearness  of  differentiation.  It  is  also 
easier  to  cut  thin  sections  from  paraffin  blocks  than  from  those 
prepared  with  celloidin. 

When  staining  tissue  sections  for  bacteria,  it  is  most  convenient 
to  carry  out  the  process  with  the  section  attached  to  a  slide.  For 
celloidin  sections  this  may  be  accomplished  by  means  of  ether  vapor. 
For  paraffin  sections  it  is  necessary  to  cover  the  slide  with  an 
extremely  thin  layer  of  a  filtered  mixture  of  equal  quantities  of 
egg  albumin  and  glycerin,  to  which  a  small  crystal  of  camphor  or 
a  drop  or  two  of  carbolic  acid  has  been  added.  The  sections  are 
then  floated  upon  a  slide  so  prepared,  and  set  away  in  the  thermostat 
for  four  or  five  hours. 

LQEFFLER'S  METHOD.58 — Stain  in  alcoholic  methylene-blue  solution  five  to 
fifteen  minutes,  or  in  Loeffler's  alkaline  methylene-blue  solution  one  to  twenty- 
four  hours. 

Wash  in  one  to  one-thousand  acetic  acid  solution  for  about  ten  seconds. 

Treat  with  absolute  alcohol  by  pouring  the  alcohol  over  the  preparation 
for  ten  to  twenty  seconds. 

Clear  with  xylol. 

Mount  in  balsam. 

31  For  details  of  such  work  reference  should  be  had  to  the  standard  textbooks 
on  pathological  technique,  notably  the  very  excellent  one  of  Mallory  and  Wright. 
**Loefiler}  Mitt.  a.  d.  kais.  Gesundheitsamt,  ii,  1884. 


130  BIOLOGY  AND  TECHNIQUE 

When  celloidin  sections  are  stained  in  this  way  ninety-five  per  cent  alcohol 
should  be  substituted  for  the  absolute.  A  number  of  other  staining  solutions 
may  be  used  in  the  same  way,  aqueous  fuchsin  or  aqueous  gentian-violet 
yielding  good  result. 

Nicolle  advises  the  use  of  a  ten  per  cent  aqueous  solution  of 
tannic  acid  for  a  few  seconds  after  washing  with  the  acetic  acid. 

Sections  may  also  be  stained  by  placing  them  over  night  into  a 
dilute  Giemsa  solution  (one  drop  to  each  c.c.  of  distilled  water). 
When  so  stained  the  sections  must  not  be  run  through  the  weaker 
alcohols  but  must  be  rapidly  differentiated  in  absolute  alcohol. 

METHOD  OF  STAINING  GRAM-POSITIVE  BACTERIA  IN  TISSUE  SECTIONS. — 
Celloidin  Sections. — After  fixing  section  to  the  slide  by  pressure  with  a  filter 
paper  or  ?by  ether  vapor,  cover  with  anilin-water  gentian-violet  five  minutes. 

Pour  off  excess  of  stain  and  cover  with  Gram's  iodin  solution  for  two 
minutes.  Decolorize  with  ninety-five  per  cent  alcohol  until  no  more  color 
comes  out. 

Stain  quickly  with  eosin-alcohol  (ninety-five  per  cent  alcohol  to  which 
enough  eosin  has  been  added  to  give  a  transparent  pink  color;  about  1:15). 
Clear  in  eosin-oil  of  origanum  (oil  of  origanum,  25  c.c.  and  eosin  alcohol, 
as  above,  about  3  c.c.).  Blot  and  mount  in  balsam. 

Paraffin  Sections. — Stain  with  anilin-water  gentian-violet  five  to  ten 
minutes. 

Wash  in  water. 

Cover  with  Gram's  iodin  solution  one  minute. 

Wash  in  water. 

Decolorize  with  absolute  alcohol  until  no  more  color  comes  out. 

Clear  in  xylol. 

Mount  in  balsam. 

Gram-Weigert  Method.™— (For  celloidin  sections.)— Stain  for  one-half 
hour  in  the  following  freshly  filtered  solution: 

Carmine  3.5  grams. 

Saturated  aqueous  solution  of  lithium  carbonate 100  c.c. 

Dehydrate  in  ninety-five  per  cent  alcohol. 

Stick  section  to  slide  with  ether  vapor. 

Stain  in  anilin-water  gentian- violet  for  five  to  fifteen  minutes  (or  in  a 
saturated  solution  of  aqueous  crystal  violet  diluted  with  water  one  to  ten, 
five  to  fifteen  minutes). 

Wash  in  physiological  salt  solution. 

Cover  with  Gram's  iodin  solution  one  to  two  minutes. 

Wash  in  water  and  blot. 


39  Weigert,  Fortschr.  d,  Med.,  v,  1887, 


MICROSCOPIC  STUDY  AND  STAINING  131 

Decolorize  with  anilin  oil  until  no  more  color  comes  off. 
This  both  decolorizes  and  dehydrates. 
Treat  with  xylol.     Mount  in  balsam. 

METHOD  FOR  GRAM  POSITIVE  AND  GRAM  NEGATIVE  ORGANISMS  IN  TISSUES, 
RECOMMENDED  BY  PAPPENHEIMER. 

Fixation:  Zenker  preferred.    Formalin  or  alcohol. 
Paraffin  sections,  5  mu  or  less. 
Staining : 

1.  Stirling's  Gentian  violet — 5  minutes. 

2.  Gram's  iodin — 1  minute. 

3.  Aniline  oil — or  aniline  oil — xylol.     Decolorize  to  pale  violet. 

4.  100%  ale.     Few  seconds  only. 

5.  Distilled  water. 

6.  Aqueous  saffranin.     l/2%  30  seconds. 

7.  Distilled  water. 

8.  Blot.     100%  ale.     Few  seconds  only. 

9.  Clear  in  xylol. 

GRAM-WEIGERT    METHOD    (for    paraffin    sections). — Preferably    Zenker's 
fixation : 

1.  Stain  sections  lightly  in  alum-hematoxylin. 

2.  Wash  in  running  water. 

3.  Four  per  cent  aqueous  solution  soluble  in  water,  five  minutes  to  one- 
half  hour. 

4.  Wash  in  water. 

5.  Aniline  methyl-violet,  one-half  to  one  hour. 

6.  Wash  off  with  water. 

7.  LugoFs  solution,  one  to  two  minutes. 

8.  Wash  off  with  water. 

9.  Blot  with  filter-paper  and  dehydrate  and  clear  in  several  changes  of 
aniline  and  xylol,  equal  parts,  or  in  aniline  oil  alone. 

10.  Wash  off  with  xylol. 

11.  Mount  in  xylol-colophonium. 

MALLORY'S   EOSIN   AND  METHYLENE-BLUE    STAIN. — For   paraffin  sections 
after  Zenker's  fixation  only: 

1.  Stain  paraffin  sections  in  a  5  per  cent  aqueous  solution  of  eosin  for 
twenty  minutes  or  longer.     Sometimes  it  is  advisable  to  get  a  deeper  eosin 
stain  by  placing  the  sections  in  the  paraffin  oven  for  fifteen  to  twenty  minutes. 

2.  Wash  in  water  to  get  rid  of  excess  of  eosin. 

3.  Stain  in  Unna's  alkaline  methylene-blue  solution,  diluted  ^4  or  5  with 
water,  for  ten  or  fifteen  minutes. 

4.  Wash  in  water. 

5.  Differentiate  and  dehydrate  in  a  cUsh  of  95  per  cent  alcohol,  to  which 


132  BIOLOGY  AND  TECHNIQUE 

has  been  added  a  few  drops  of  a  10  per  cent  solution  of  colophonium  in 
absolute  alcohol,  keeping  the  section  in  constant  motion,  so  that  the  decoloriza- 
tion  shall  be  uniform.  Control  the  result  under  the  microscope.  When  the 
pink  color  has  returned  to  the  section  and  the  nuclei  are  still  a  deep  blue, 
finish  the  dehydration  quickly  with  absolute  alcohol. 

6.  Xylol. 

7.  Xylol  balsam. 

METHOD  OF  STAINING  FOR  TUBERCLE  BACILLI  IN  SECTIONS.*" — Paraffin 
Sections. — Stain  in  carbol-fuchsin  solution  hot  for  five  minutes  (or  better 
cold,  for  twenty-four  hours). 

Wash  in  water. 

Decolorize  and  countorstain  in  Gabbet's  methylene-blue  sulphuric  acid 
mixture  for  one  minute. 

Wash  in  water. 

Dehydrate  in  absolute  alcohol. 

Clear  in  xylol. 

Mount  in  balsam. 

Celloidin  Sections.41 — Stain  lightly  in  alum  hematoxylin. 

Wash  in  water. 

Dehydrate  in  ninety-five  per  cent  alcohol. 

Attach  the  slide  by  ether  vapor. 

Stain  with  steaming  carbol-fuchsin  two  to  five  minutes. 

Wash  in  water. 

Wash  with  Orth's  acid  alcohol  (alcohol  ninety  per  cent.,  99  c.c.;  cone. 
HC1,  1  c.c.)  one-half  to  one  minute. 

Wash  in  water  several  changes. 

Treat  with  ninety-five  per  cent  alcohol  until  red  color  is  entirely  gone. 

Blot  and  cover  with  xylol  until  clear.    Mount  in  balsam. 

METHOD  OF  STAINING  ACTINOMYCES  IN  SECTIONS. — Mallory's  Method.412 
— 1.  Stain  deeply  in  saturated  aqueous  eosin  ten  minutes. 

2.  Wash  in  water. 

3.  Anilin  gentian-violet  two  to  five  minutes. 

4.  Wash  in  normal  saline  solution. 

5.  Weigert's  iodin  solution    (iodin  1,  KI   2,  and  water  100   parts)    one 
minute. 

6.  Wash  in  water  and  blot. 

7.  Clear  in  anilin  oil. 

8.  Xylol  several  changes. 

9.  Mount  in  balsam. 


^Mallory  and  Wright, 

41  After  Mallory  and   Wright. 

"Mallory  and  Wright,  "Pathol.  Tech.,"  1904. 


CHAPTER  VII 

THE  PREPARATION  OF  CULTURE  MEDIA 

GENERAL    TECHNIQUE 

THE  successful  cultivation  of  bacteria  upon  artificial  media  re- 
quires the  establishment  of  an  environment  which  shall  be  suitable 
in  regard  to  the  presence  of  assimilable  nutritive  material,  moisture, 
and  osmotic  relations.  These  requirements  are  fulfilled  in  the  com- 
position of  the  nutrient  media  described  in  another  section,  media 
which  are  to  some  extent  varied  according  to  the  special  require- 
ments of  the  bacteria  which  are  to  be  cultivated.  If  cultivation, 
furthermore,  is  to  have  any  value  for  scientific  study  of  individual 
species,  it  is  necessary  to  obtain  these  species  free  from  other  varie- 
ties of  microorganisms,  that  is,  in  pure  culture,  and  to  protect  such 
cultures  continuously  from  contamination  with  the  other  innumer- 
able species  which  are  everywhere  present. 

The  technique  which  is  employed  for  these  purposes  has  been 
gradually  evolved  from  the  methods  originally  devised  by  Pasteur, 
Koch,  Cohn,  and  others. 

Bacterial  cultivation  is  carried  out  in  glassware  of  varied  con- 
struction, the  forms  most  commonly  employed  being  test  tubes  of 
various  sizes,  Erlenmeyer  flasks,  the  common  Florence  flasks,  and 
Petri  dishes.  All  glassware,  of  course,  must  be  thoroughly  cleansed 
before  being  used. 

Preparation  of  Glassware. — The  cleansing  of  glassware  may  be 
accomplished  by  any  one  of  a  number  of  methods.  New  glassware 
may  be  immersed  in  a  one  per  cent  solution  of  hydrochloric  or  nitric 
acid  in  order  to  remove  the  free  alkali  which  is  occasionally  present 
on  such  glass.  It  is  then  transferred  to  a  one  per  cent  sodium 
hydrate  solution  for  a  few  hours,  and  following  this  is  washed  in 
hot  running  water. 

In  the  case  of  old  glassware  which  has  contained  culture  media, 
sterilization  in  the  autoclave  is  first  carried  out,  then  the  glassware 
is  boiled  in  five  per  cent  soda  solution  or  in  soapsuds.  After  this, 

133 


134  BIOLOGY  AND  TECHNIQUE 

thorough  mechanical  cleansing  is  practiced,  and  the  glassware  may 
be  treated  by  acid  and  alkali  followed  by  running  water,  as  given 
above.  These  last  steps,  however,  are  not  essential,  thorough  wash- 
ing in  hot  water  after  the  soapsuds  or  soda  solution*  being  usually 
sufficient  to  yield  good  results.  Other  workers  have  recommended 
immersion  of  the  glassware  after  mechanical  cleansing  in  five  per 
cent  to  ten  per  cent  potassium  bichromate  solution  in  twenty-five  per 
cent  sulphuric  acid.  This  is  followed  by  thorough  washing  in  hot 
running  water,  and  drying. 

Clean  flasks  and  test  tubes  are  then  stoppered  with  cotton,  which 
has  been  found  to  be  a  convenient  and  efficient  seal  against  the 
bacteria  of  the  air,  catching  them  in  the  meshes  of  the  fibers  as  in 
a  filter.  The  technique  of  the  stoppering  or  plugging  of  glass  recep- 
tacles is  important,  in  that,  when  poorly 'plugged,  sterility  is  not 
safeguarded,  and  the  purpose  of  culture  study  is  defeated. 

In  almost  all  laboratories  in  this  country  non-absorbent  cotton 
or  "cotton  batting"  is  used  for  the  plug.  In  a  few  of  the  German 
laboratories,  the  absorbent  variety  is  employed.  The  disadvantages 
of  the  latter,  especially  in  the  case  of  fluid  media,  are  obvious.  The 
plugs  should  fit  snugly,  but  not  so  tightly  that  force  is  necessary 
to  remove  them.  Care  should  be  taken,  furthermore,  that  no  creases 
are  left  between  the  surface  of  the  glass  and  the  periphery  of  the 
plug;  for  these,  if  present,  may  serve  as  channels  for  the  entrance 
of  bacteria.  The  plugging  itself  is  carried  out  by  tearing  a  small 
piece  of  cotton,  about  2X2  inches,  from  the  roll,  folding  over  one 
of  its  corners,  and,  applying  the  smooth  end  of  a  glass  rod  to  the 
folded  portion,  gently  pushing  it  into  the  mouth  of  the  tube. 

After  plugging  and  before  media  are  introduced  into  the  tubes 
and  flasks,  these  should  be  sterilized.  This  is  best  done  in  one  of 
the  "hot-air  sterilizers,"  by  exposing  the  tubes  for  one  hour  to  a 
temperature  of  150°  C.  If  greater  speed  is  desired  exposure  to 
180°  to  190°  C.  for  half  an  hour  is  usually  safe.  If  by  mistake, 
however,  the  temperature  is  allowed  to  rise  above  200°  C.,  a  brown- 
ing of  the  cotton  plugs  occurs  and  the  glassware  is  apt  to  be  stained 
by  the  burning  of  the  fat  and  other  organic  material  derived  from 
the  cotton.  Petri  dishes  after  cleansing  are  fitted  together,  and  then 
sterilized  in  the  hot-air  chamber  at  150°  C.  for  one  hour. 

Glassware  so  prepared  is  ready  for  the  reception  of  media. 

Ingredients  of  Culture  Media.— The  food  requirements  of  bac- 
teria have  been  discussed  in  another  section.  From  what  has  there 


THE   PREPARATION   OF  CULTURE   MEDIA  135 

been  said,  it  is  apparent  that  artificial  culture  media  must,  to  a 
certain  extent,  be  adjusted  to  the  peculiarities  of  individual  bacteria. 
In  the  cases  of  the  more  strictly  parasitic  microorganisms  growth 
can  be  obtained  only  by  the  most  rigid  observance  of  special  require- 
ments. For  the  large  majority  of  pathogenic  bacteria,  however, 
routine  or  standard  media  may  be  employed,  which,  while  slightly 
more  favorable  for  one  species  than  for  another,  are  sufficiently 
general  in  their  composition  to  permit  the  growth  of  all  but  the 
most  fastidious  varieties. 

The  basis  of  many  of  our  common  media  is  formed  by  the  soluble 
constituents  of  meat.  These  substances  are  best  obtained  by 
macerating  500  grams  of  lean  beef  in  1,000  c.c.  of  distilled  water. 
The  mixture  is  allowed  to  infuse  in  the  ice  chest  over  night,  and 
then  strained  through  cheese-cloth.  To  this  infusion  are  added  the 
other  required  constituents  in  the  manner  given  in  the  detailed  in- 
structions below.  The  soluble  constituents  of  meat,  however,  may 
also  be  procured  in  a  simpler  way  by  the  use  of  the  commercial 
meat  extracts,  such  as  that  of  Liebig.  These  extracts  are  dissolved 
in  quantities  of  five  grams  to  the  liter,  and  other  constituents  are 
added  to  this  nutrient  basis. 

Though  simpler  to  make,  the  meat-extract  media  are  less  favor- 
able for  the  cultivation  of  the  more  delicate  organisms  than  are  the 
media  made  directly  from  fresh  meat.  Nevertheless,  they  suffice 
for  the  cultivation  of  the  large  majority  of  the  more  saprophytic 
path'o genie  microorganisms  and  hold  an  important  place  in  labora- 
tory technique. 

The  ingredients  and  methods  used  in  various  laboratories  in  the 
preparation  of  such  standard  media  should  be,  as  much  as  possible, 
uniform,  in  order  that  confusion  in  results  may  be  avoided;  for, 
as  is  well  known,  the  biological  characteristics  of  one  and  the  same 
bacterial  species  may  vary  considerably  if  grown  on  media  differing 
in  their  composition. 

A  committee  of  the  American  Public  Health  Association,1  ap- 
pointed in  1897  for  the  sake  of  standardizing  the  methods  of  prepara- 
tion of  media,  recommended  that  the  following  rules  should  govern 
the  choice  of  ingredients : 

1.  Distilled  water  should  be  used  in  all  cases.     (This  is  not  neces- 

'Rep.  Com.  of  Amer.  Bact,  to  Com.  of  Amer.  Pub,  Health  Assn.  Meeting, 
Philadelphia,  Sept.,  1897, 


136  BIOLOGY  AND  TECHNIQUE 

sary  for  routine  laboratory  media  if  the  constitution  of  the  tap 
water  is  known  and  controlled  from  time  to  time.) 

2.  The  meat  used  should  be  fresh,  lean  beef  (when  veal  or  chicken 
is  substituted  the  change  should  be  stated). 

3.  The  pepton  used  should  be  Witte's  pepton,  dry,  made  from 
meat.2 

4.  Only  C.  P.  NaCl  should  be  used. 

5.  For  alkalinizing  C.  P.  sodium  hydrate  should  be  used  in  normal 
solutions. 

6.  For  acidification  C.  P.  hydrochloric  acid  in  normal  solution 
should  be  used. 

7.  When  glycerin  is  used,  this  should  be  of  the  neutral  redistilled 
variety. 

8.  The   agar-agar   employed   should  be   of  the   finest   grade    of 
commercial  thread  agar. 

9.  The  gelatin  should  be  the  commercial  sheet  gelatin  washed  as 
free  as  possible  of  acid  and  impurities. 

10.  Chemicals  and  carbohydrates  which  are  used  should  be  as 
nearly  chemically  pure  as  possible. 

Titration  of  Media. — Next  in  importance  to  the  actual  composi- 
tion of  media  is  the  adjustment  of  their  reaction.  Bacteria  are 
highly  susceptible  to  varations  in  the  acidity  and  alkalinity  of  media, 
excessive  degrees  of  either  may  completely  inhibit  development  or 
moderate  varations  may  lead  to  marked  modifications  of  cultural 
characteristics.  It  is  necessary,  therefore,  to  adjust  the  reaction 
both  f  orx  the  sake  of  favoring  growth  and  in  order  to  insure  uni- 
formity of  growth  characters.  This  is  accomplished  by  titration 
which  is  best  carried  out  according  to  the  recommendations  of  the 
committee  mentioned  above. 

OLDER  METHOD  OF  DIRECT  TITRATION.— The  color  indicator  employed  for 
the  titration  is  a  five-tenths  per  cent  solution  of  phenolphthalein  in  fifty  per 
cent  alcohol..  The  chief  advantage  of  this  indicator  over  others  is  due  to 
the  fact  that  it  indicates  the  presence  of  organic  acid  and  acid  compounds 
in  its  reaction.  For  actual  titration  -^L  (_!_  normal)  solutions  of  sodium 
hydrate  or  of  hydrochloric  acid  are  used.  Since  media  in  the  process  of 
preparation  are  usually  acid,  the  NaOH  solution  is  the  one  most  frequently 


-  Since  the  Trar  many  domestic  peptons  have  been  introduced  and  can  be  used 
with  success. 


THE  PREPARATION  OF  CULTURE   MEDIA  137 

needed.  Five  c.c.  of  the  medium  to  be  tested  is  measured  accurately  in  a 
carefully  washed  pipette  and  transferred  into  a  porcelain  evaporating'  dish. 
To  this  are  added  45  c.c.  of  distilled  water.  The  mixture  is  thoroughly  boiled 
for  three  minutes  over  a  free  flame.  The  boiling  drives  off.  C02,  giving  the 
true  neutral  point,  and  approximates  the  conditions  prevailing  during  the 
further  sterilization  of  the  medium  from  which  the  5  c.c.  have  been  taken. 
After  boiling,  1  c.c.  of  the  phenolphthalein  is  added.  If  the  medium  is  acid, 
no  color  is  present;  if  alkaline,  a  pink  or  red  color  appears.  The  ^~  alkali 
or  acid  solution  is  allowed  to  drop  into  the  dish  from  a  graduated  burette. 
When  the  neutral  point  is  approached  in  an  acid  solution,  each  drop  of 
sodium  hydrate  added  brings  forth  at  first  a  deep  red,  which,  however,  upon 
slight  stirring  with  a  clean  rod,  completely  disappears.3  The  end  reaction 
is  reached  when  a  faint  but  clear  and  distinct  pink  color  remains  in  the  fluid 
after  stirring. 

When  titrating  alkaline  media,  the  addition  of  the  phenolphthalein 
produces  a  red  color  in  the  hot  medium  which  gradually  fades  upon  the 
addition  of  ^  HC1,  becoming  colorless  at  the  end  point  of  titration.  Titra- 
tion  should  be  done  quickly  and  in  a  hot  solution.  From  the  result  of  the 
titration  the  computation  for  the  neutralization  of  the  entire  bulk  of  the 
medium  can  be  made  by  a  simple  arithmetical  process  as  illustrated  in  the 
following  example: 

Let  us  suppose  that  we  have  used: 

2.5  c.c.  of^L_NaOH  to  neutralize  5  c.c,  of  the  medium, 

then  2.5  c.c.  of  *  NaOH  will  neutralize      100  c.c.       "  " 

and     25  c.c.  of  ^  NaOH  will  neutralize  1,000  c.c.,  or  one  liter. 

The  adjustment  of  the  reaction  of  media  is  largely  determined 
by  the  particular  uses  for  which  the  media  are  designed.  For 
examinations  in  the  practice  of  sanitation,  such  as  analyses  of  water, 
ice,  and  milk,  etc.,  the  American  Public  Health  Association  recom- 
mends a  standard  reaction  of  -(-  1  per  cent  (the  plus  sign  is  used 
to  indicate  acidity,  the  minus  alkalinity ;  +  1  per  cent  is  the  expres- 
sion used  to  indicate  that  one  per  cent  of  ^  sodium  hydrate  solution 
would  be  required  to  neutralize  the  medium  or  10  c.c.  to  the  liter). 
For  general  work  with  pathogenic  bacteria,  the  most  favorable 
reaction  for  routine,  media  is  slight  alkalinity,  neutrality,  or  an 
acidity  not  exceeding  -f-  1  per  cent. 

3  See  standard  textbooks  on  volumetric  analysis. 


138  BIOLOGY  AND  TECHNIQUE 

THE  COLORIMETRIC  METHOD  OF  TITRATION 

The  method  given  above  is  not  an  accurate  one  because,  in  thje 
titration  of  culture  media,  we  are  dealing  with  solutions  which 
contain  considerable  quantities  of  materials,  such  as  peptone,  pro- 
teins, phosphates,  etc.,  which  have  an  action  which  is  spoken  of 
technically  as  that  of  a  "buffer."  This  term  signifies  the  power  of 
these  substances  to  oppose  changes  in  reaction.  The  degree  of 
"buffer"  action,  as  shown  by  a  number  of  writers,  but  recently  in 
connection  with  bacteriological  work  particularly  by  Clark  and 
Lubs,4  is  proportionate  to  the  concentration  of  the  constituents,  and 
the  consequence  of  the  action  is  that  volumetrically  proportionate 
amounts  of  acid  or  alkali  added  to  such  solutions  do  not  change  the 
reaction  in  the  same  proportions.  Curves  found  in  the  paper  of 
Clark  and  Lubs  cited  above,  will  make  this  sufficiently  clear.  In 
consequence,  when  we  titrate  5  cubic  centimeters  of  the  medium 
as  in  the  old  method,  to  the  neutral  point  of  phenolphthalein,  we 
could  bring  the  entire  media  to  the  neutral  point  of  phenolphthalein 
by  adding  proportionate  amounts  of  acid  or  alkali,  but  we  cannot 
foretell  the  final  hydrogen  ion  concentration  attained  in  the  medium 
by  adding  fractions  of  this  total  amount.  It  is  plain,  therefore,  that 
to  make  an  accurate  adjustment  of  media  it  would  be  better  to 
apply  either  an  electrode  method  or  a  colorimetric  method  to  the 
medium,  adjusting  to  a  standard  of  known  hydrogen  ion  concen- 
tration. The  potentiometer  methods  have  been  considerably  simpli- 
fied, and  have  been  applied  by  men  like  Clark  and  Lubs  and  others 
who  are  thoroughly  versed  in  the  handling  cf  such  instruments. 
For  routine  laboratory  work  colorimetric  methods  have  been  in- 
troduced which,  as  used  by  others  and  by  us,  have  checked  up  quite 
accurately  with  potentiometer  measurements. 

Before  describing  the  method  at  present  in  use,  it  will  be  best 
to  say  a  few  words  about  the  nomenclature  used  at  present  for  the 
expression  of  hydrogen  ion  concentrations. 

THE  MEANING  OF  PH.  The  hydrogen  ion  concentration  of  pure  water 
is  0.0000001.  This  is  more  simply  expressed  as  1X10~7.  Since, 
in  pure  water,  hydroxyl  ions  are  equal  in  concentration,  to  the  hydro- 
gen ions,  and  the  hydroxyl  ions  are  also  1 X  10~7,  and  this  is  the  neutral 


4  Clark  and  Lubs,  Jour,  of  Bacter.,  2,  1917,  1,  109,  191. 


THE   PREPARATION   OF  CULTURE   MEDIA  139 

point.  Solutions  in  which  the  hydrogen  ion  concentration  is  less  than 
1X10~7  (and  the  hydroxyl,  therefore,  more  than  1X10~7)  are  alkalin, 
and  vice  versa.  This  method  of  expression  offers  certain  difficulties 
to  the  plotting  of  curves,  and  it  has  been  found  simpler  for  such  purposes 
to  plot  curves  according  to  the  logarithms  of  the  expressions  used 
above.  Sorensen 5  has  initiated  a  method  of  doing  this,  by  intro- 
ducing the  symbol  PH  which  signifies  the  logarithm  of  the  reciprocal 
of  the  hydrogen  ion  concentration,  expressed  as  above,  thus,  PH  equals 

tog  of -L. 

Thus,  if: 

H  =  2X10-4, 


5>2X10-4 
=  log  5000  =  3.7. 

Another  way  of  getting  at  this  is  as  follows: 
Supposing  that, 

[H]  =  2X10-6, 

Pj?/  =  log.  of  reciprocal, 
or, 


Now, 

log.  1  =  0, 

log.  2  =  0.3, 
log.  10~6=-6. 
Therefore  : 


or, 

P7/  =  0-[+0.3-6]     or     5.7. 

The  following  table  gives  the  relationship  of  the  old  method  of 
expression  to  the  new: 


5  Sorensen,  Biochem.  Zeit.,  1909,  21,  131,  201. 


140 


BIOLOGY  AND  TECHNIQUE 
HYDROGEN  ION  CONCENTRATION     PH  VALUE 


ixio-1 

1 

IXIO-2 

2 

IXIO-3 

3 

IXIO-4 

4 

1X10~5 

5 

ixio-6 

6 

IXIO"7 

7 

1X10~8 

8 

ixio-9 

9 

1X10~10 

10 

Indicators  are  substances,  usually  weak  organic  acids  or 
which  change  in  color  when  subjected  to  changes  of  reaction,  that  is, 
to  changes  of  hydrogen  or  hydroxyl  ion  concentrations.  A  table 
which  we  take  from  a  pamphlet  prepared  by  the  Army  Medical  School, 
will  give  the  color  reactions  at  various  P#  measurements  for  a  number 
of  common  indicators. 

COLOR  REACTIONS  IN  SOLUTIONS  OF  DIFFERENT  HYDROGEN  ION  CONCENTRATION  (2) 


True  acidity 

10~3 

10~4 

10~5 

10~6 

10~7 

10~8 

10~9 

10-io 

Methyl  orange 

Rose 
red 

Orange 
red 

Yellow 



<  — 

<- 

«- 

^ 

Rosolic  acid 

Yellow 

- 

-» 

-> 

Rose 

Red 

- 

- 

Congo  red 

Blue 

Violet 

Reddish 
violet 

Orange 

Orange 
red 

«- 

*- 

*^ 

Litmus 

Red 

-* 

-> 

Reddish 
violet 

Violet 

Blue 

'*- 

<— 

Phenolphthalein 

Color- 
less 

-> 

7» 

r» 

-» 

-* 

Red 

<  — 

Clark  and  Lubs  particularly  have  been  responsible  for  introducing 
the  method  at  present  in  use  in  bacteriological  laboratories,  based 
upon  the  preparation  of  solutions  which  can  be  used  as  standards  for 
the  colorimetric  measurements  of  culture  media.  The  solutions  must 
be  very  carefully  prepared,  and  should,  whenever  possible,  be  con- 
trolled by  potentiometer  measurements;  however,  with  proper  care 


THE   PREPARATION   OF  CULTURE   MEDIA 


141 


and  the  use  of  pure  substances  by  a  reliable  worker,  this  may,  with 
reasonable  safety,  be  omitted.  The  principle  of  making  the  dilutions 
is  that  carefully  measured  amounts  of  molecular  solutions  of  acids 
and  alkalis  are  mixed  in  series  so  that  each  successive  tube  shall  con- 
tain a  definite  hydrogen  ion  concentration.  These  tubes  are  the 
standard.  Clark  and  Lubs,  in  the  first  of  the  series  of  papers  noted  above, 
cite  tables  of  mixtures  for  such  purposes,  with  a  range  of  PH  extending, 
from  1  to  10.  For  the  exact  composition  of  all  of  these  mixtures, 
the  reader  is  referred  to  the  original  paper  of  Clark  and  Lubs,  page  25. 
For  the  routine  of  the  ordinary  laboratory  bacteriology,  only  the 
fourth  section  of  this  table  is  necessary.  This  is  as  follows: 

KH2PO4— NaOH 


5.8 

50  c.c.  M/5  KH2PO4 

3.  72  c.c.  M/5  NaOH 

Dilute  to  200  c.c. 

6.0 

50  c.c.  M/5  KH2PO4 

5.  70  c.c.  M/5  NaOH 

Dilute  to  200  c.c. 

6.2 

50  c.c.  M/5  KH2PO4 

8.  60  c.c.  M/5  NaOH 

Dilute  to  200  c.c. 

6.4 

50  c.c.  M/5  KH2PO4 

12.  60  c.c.  M/5  NaOH 

Dilute  to  200  c.c. 

6.6 

50  c.c.  M/5  KH2PO4 

17.  80  c.c.  M/5  NaOH 

Dilute  to  200  c.c. 

6.8 

50  c.c.  M/5  KH2PO4 

23.  65  c.c.  M/5  NaOH 

Dilute  to  200  c.c. 

7.0 

50  c.c.  M/5  KH2PO4 

29.  63  c.c.  M/5  NaOH 

Dilute  to  200  c.c. 

7.2 

50  c.c.  M/5  KH2PO4 

35.  00  c.c.  M/5  NaOH 

Dilute  to  200  c.c. 

7.4 

50  c.c.  M/5  KH2PO4 

39.50  c.c.  M/5  NaOH 

Dilute  to  200  c.c. 

7.6 

50  c.c.  M/5  KH2PO4 

42.  80  c.c.  M/5  NaOH 

Dilute  to  200  c.c. 

7.8 

50  c.c.  M/5  KH2PO4 

45.  20  c.c.  M/5  NaOH 

Dilute  to  200  c.c. 

8.0 

50  c.c.  M/5  KH2PO4 

46.80  c.c.  M/5  NaOH 

Dilute  to  200  c.c. 

The  method  of  procedure  in  using  these  solutions  is  as  follows: 
From  the  solutions  made  above  a  colorimetric  scale  is  prepared.  All 
glassware  must  be  very  carefully  cleansed,  and  it  should  be  remem- 
bered that  some  of  the  cheaper  glassware  obtained  in  laboratories 
at  the  present  time  often  seems  to  give  off  considerable  amounts  of 
alkali.  Whatever  method  of  cleaning  is  used,  the  final  thorough 
rinsing  must  be  done  thoroughly  with  redistilled  water.  10  c.c.  each  of 
the  respective  series  of  standard  mixtures  is  placed  into  each  test 
tube,  and  to  it  10  drops  of  the  indicator  are  added.  For  the  range 
from  6.8  to  8.4,  which  is  sufficient  for  all  ordinary  pathogenic  work, 
the  indicator  used  is  phenol-sulfon-phthalein,  or  phenol  red  in  con- 
centration of  0.02  per  cent  aqueous  solution.  If  ranges  from  6  to  7.6 
are  desired,  brom-thymol  blue  in  a  concentration  of  0.04  per  cent 
may  be  used,  and  when  ranges  just  above  8  are  desired,  cresol  red 
in  concentration  of  0.02  per  cent  is  recommended. 


142 


BIOLOGY  AND  TECHNIQUE 


A  series  of  tubes  so  prepared,  each  of  which  contains  10  c.c.  of 
each  of  the  graded  mixtures,  with  indicator  added,  represents  a 
colorimetric  scale  against  which  the  media  can  be  standardized. 

This  standardization  is  now  carried  out  as  follows,  and  can  be 
utilized  for  any  media  which  are  not  too  highly  colored  and  not  turbid: 
Into  a  thoroughly  cleaned  test  tube  2  c.c.  of  the  medium  are  measured 
and  this  diluted  with  8  c.c.  of  redistilled  water.  10  drops  of  indicator 
are  then  added,  and  after  thoroughly  mixing,  a  color  reading  is  taken 
against  the  scale.  If  the  reaction  is  too  acid,  as  is  usually  the  case, 

N 
add  =77  NaOH  from  a  burette  or  graduated  pipette,  a  drop  at  a  time, 

until  the  color  matches  that  of  the  standard  tube.  By  calculating 
from  the  amount  of  weak  alkali  added,  the  total  quantity  of  media 

N 
is  then  brought  to  the  desired  P#  with  —  NaOH.     In  the  titration  of 

agar,  Clark  and  Lubs  recommend  that  the  broth  be  titrated  and 
adjusted  before  the  addition  of  the  agar,  in  order  to  avoid  possible 
colloidal  changes  between  the  agar  and  the  indicator,  and  that  the 
agar  be  added  after  the  adjustment  is  made.  It  is  possible,  however, 
without  excessive  error,  to  carry  out  the  titration  of  media  containing 
agar  in  the  same  way  as  outlined  above,  adding  cold  water  to  the 
hot  agar,  and  making  the  comparison  at  once  at  a  temperature  of 
35°  to  40°  before  the  mixture  has  jelled.  In  solutions  like  bacterial 
media,  which  haw  a  certain  amount  of  color  or  some  turbidity,  a 
so-called  " comparator "  may  be  used  in  the  form  of  a  wooden  box 
painted  black  with  four  holes  for  test  tubes  and  a  slit  in  front  and 
behind,  so  that  it  can  be  looked  through  against  a  source  of  light. 
The  arrangement  of  this  is  given  in  the  following  cut: 


LIGHT  SOURCE 


EYE 


Arrangement  for  Reading  Titration. 


THE   PREPARATION   OF  CULTURE   MEDIA  143 

Methods  of  Clearing  Media. — Clearing  with  Eggs. — When  culture 
media"  are  prepared  from  substances  containing  no  coagulable  pro- 
tein, it  is  often  necessary,  for  purposes  of  clearing,  to  add  the  whites 
of  eggs,  and  then  to  heat  for  forty-five  minutes  in  the  Arnold 
sterilizer.  In  the  following  detailed  descriptions,  the  direction 
"clear  with  egg"  has  been  given  whenever  such  a  step  is  deemed 
necessary.  The  exact  technique  of  such  a  procedure  is  as  follows: 

In  a  small  pot  or  pan,  the  whites  of  several  eggs  (one  or  two 
eggs  to  each  liter  of  medium)  are  beaten  up  thoroughly  with  a  little 
water  (20  c.c.).  This  egg  white  is  then  poured  into  the  medium, 
which,  if  hot,  as  in  the  case  of  melted  agar  or  gelatin,  must  first 
be  cooled  to  about  50°  to  55°  C.  The  mixture  is  then  thoroughly 
shaken  and  steamed  in  the  Arnold  sterilizer  for  thirty  minutes.  At 
the  end  of  this  time,  the  flask  containing  the  medium  is  removed  from 
the  sterilizer  and  thoroughly  shaken  so  as  completely  to  break  up 
the  coagulum  which  has  formed.  It  is  then  replaced  and  allowed 
to  steam  for  another  fifteen  minutes.  At  the  end  of  this  time  the 
medium  between  the  coagula  should  be  clear.  It  is  now  ready  for 
filtration  through  cotton. 

Filtering  Media  through  Cotton. — The  filtration  of  media  after 
clearing,  either  by  the  addition  of  eggs  or  by  the  coagulation  of  the 
proteins  originally  contained  in  it,  is  best  done  through  absorbent 
cotton.  A  small  spiral,  improvised  of  copper  wire  is  placed  as  a 
support  in  the  bottom  of  a  large  glass  funnel.  A  square  piece  of 
absorbent  cotton  is  then  split  horizontally  giving  two  squares  of 
equal  size.  Ragged  edges  and  incisures  should  be  avoided.  These 
two  layers  of  cotton  are  then  placed  in  the  funnel,  one  piece  above 
the  other  in  such  a  way  that  the  direction  of  the  fibers  of  the  two 
layers  is  at  right  angles  one  to  the  other.  They  are  then  gently 
depressed  "into  the  filter  with  the  closed  fist.  The  edges  of  the 
cotton  are  made  to  adhere  to  the  sides  of  the  funnel  by  allowing 
a  thin  stream  of  tap  water  to  run  over  them,  while  smoothing  them 
against  the  glass  with  the  hand. 

The  medium,  when  poured  into  such  a  filter,  should  be  poured 
along  a  glass  rod  at  first,  to  avoid  running  down  the  sides  or  burst- 
ing the  filter.  After  filtration  has  begun,  the  filter  should  be  kept 
as  full  as  possible.  The  first  liter  or  so  which  comes  through  may 
not  be  clear,  but  the  filter  gains  in  efficiency  as  the  coagulum  settles 
into  the  fibers  of  the  cotton,  and  the  first  yield  may  be  sent  through 
a  second  time.  Filtration  of  agar  or  gelatin  is  best  done  in  a  warm 


144  BIOLOGY  AND  TECHNIQUE 

room  with  windows  and  doors  closed,  and  the  filter  covered  with 
a  lid,  to  avoid  too  rapid  cooling.  The  funnel  and  filter  should  be 
warmed  just  before  use. 

Filtering  through  Paper. — Many  media  may  be  efficiently  cleared 
by  filtration  through  close  filter  paper  without  the  aid  of  coagula. 

The  Tubing  of  Media. — Most  of  the  media  described  in  the  fore- 
going section  are  used  in  test  tubes.  In  order  to  fill  these  tubes, 
the  media  are  best  poured  into  a  large  glass  funnel  to  which  a  glass 
discharging  tube  has  been  fitted  by  means  of  a  short  piece  of  rubber 
tubing.  Upon  this  is  placed  a  thumb  cock.  The  plug  is  then 
removed  from  the  test  tube  by  catching  it  between  the  small  and 
ring  fingers  of  the  right  hand  and  the  glass  outlet  is  thrust  deeply 
into  the  test  tube,  in  order  to  prevent  the  medium  from  touching 
the  upper  portion  of  the  test  tube  where  the  cotton  plug  will  be 
lodged.  About  7  to  8  c.c.  are  put  in  each  test  tube. 

Sterilization  of  Media. — By  Heat. — Media  which  contain  neither 
sugars,  gelatin,  glycerin,  nor  animal  serum  may  be  sterilized  in  the 
autoclave  at  fifteen  pounds  pressure  for  fifteen  minutes  to  half  an 
hour.  Media  which  contain  these  or  other  substances  subject  to 
injury  from  the  high  temperature,  must  be  sterilized  by  the  frac- 
tional method,  i.e.,  by  twenty  minutes'  exposure  in  the  live  steam 
sterilizer  (Arnold,  Fig.  8,  page  83)  on  each  of  three  consecutive  days. 
During  the  intervals  between  sterilizations,  they  should  be  kept  at 
room  temperature  or  in  the  incubator,  to  permit  the  germination 
of  spores  which  may  be  present.  Media  containing  animal  serum 
or  other  albuminous  solutions  which  are  to  be  sterilized  without 
coagulation,  may  be  sterilized  in  water  baths,  or  in  hot-air  chambers, 
at  temperatures  varying  from  60°  to  70°  C.,  by  the  fractional  method. 
In  such  cases  five  or  six  exposures  of  one  hour  on  succeeding  days 
should  be  employed. 

By  Filtration. — It  is  often  desirable  in  bacteriological  work  to 
free  fluid  from  bacteria.  This  is  frequently  necessary  for  the  sterili- 
zation of  blood-serum  or  exudate  fluids,  or  fOr  obtaining  toxins  free 
from  bacteria.  For  these  purposes  a  large  variety  of  filters  are  in 
use.  Those  most  commonly  employed  are  of  the  Chamberland6  or 
Berkefeld  type,  which  consist  of  hollow  candles  made  of  unglazed 
porcelain  or  diatomaceous  earth.  Both  these  types  are  made  in 
various  grades  of  fineness,  upon  which  depend  both  the  speed  of 


•  Pasteur  and  Chamberland,  Compt.  rend,  de  1'acad.  des  sci.,  1884. 


THE   PREPARATION   OF  CULTURE   MEDIA  145 

filtration  and  the  efficiency.     They  are  made  in  various  forms  and 
models,  some  of  which  are  shown  in  the  accompanying  figures.    In 


JIG.  10. — BERKEFELD  FILTER. 


FIG.  11.— REICHEL  FILTER. 

most  of  the  methods  of  filtration  commonly  employed  the  fluid  which 
is  to  be  filtered  is  sucked  through  the  walls  of  the  filter,  either  by 


146  BIOLOGY  AND  TECHNIQUE 

a  hand  suction-pump  or  by  some  form  of  vacuum-pump  attached 
to  an  ordinary  water-tap. 

The  hollow  candle-filter  may  either  be  firmly  fitted  into  a  cylin- 
drical glass  chimney  and  surrounded  by  the  fluid  which  is  to  be 
filtered,  or  else  the  candle  may  be  connected  to  the  collecting  flask 
with  sterile  tubing  and  suspended  freely  in  the  fluid.  Perfect  filters 
of  these  types  will  hold  back  any  of  the  bacteria  known  to  us  at 
present. 

Filters  before  use  must  be  sterilized.  The  candles  themselves 
are  subjected  to  150°  C.  in  the  hot-air  sterilizer  for  one  hour.  The 
glassware  and  washers  necessary  for  setting  up  the  apparatus  may 
be  sterilized  by  boiling.  In  order  that  filters  may  be  repeatedly 
used  with  good  result,  it  is  necessary  that  they  should  be  carefully 
cleaned  from  time  to  time.  This  is  best  done  in  the  following  way : 

Filters  through  which  fluids  from  living  cultures  have  passed 
are  first  sterilized  in  the  Arnold  steam  sterilizer.  Their  exterior  is 
then  carefully  cleaned  with  a  fine  brush.  Following  this  a  five- 
tenths  per  cent  solution  of  potassium  permanganate  is  passed 
through  them  and  this  again  removed  by  sucking  through  a  five 
per  cent  solution  of  bisulphite  of  soda.  This  last  is  washed  out 
by  sending  a  considerable  quantity  of  distilled  water  through  the 
filter,  which  is  then  dried  and  sterilized  by  heat. 

The  suction  necessary  for  filtration  through  these  filters  is  usually 
applied  by  means  of  the  ordinary  suction-pump  attached  to  a  run- 
ning faucet. 

Slanting  of  Media. — Solid  media  which  are  to  be  used  in  slanted 
form  in  test  tubes  should  be  inclined  on  a  ledge  (easily  improvised 
of  glass  tubing)  at  the  proper  slant,  after  the  last  sterilization. 
Agar,  the  medium  most  frequently  employed  in  this  way,  should 
be  left  in  this  position  until  hard. 

Indicators. — We  cannot  in  this  place  go  into  details  concerning 
the  theory  of  indicators.  For  this  we  may  refer  the  reader  to  the 
recent  treatise  by  Clark  on  the  "Determination  of  Hydrogen  Ions."  7 
According  to  Ostwald,  indicators  are  acids  or  bases  whose  undis- 
sociated  molecules  "have  a  different  color  from  that  of  their  dis- 
sociation products."  This  conception  has  been  somewhat  modified 
by  more  recent  work,  in  that  color  changes  are  found  to  be  associated 

''Clark,  W.  Mansfield,  Determination  of  Hydrogen  Ions,  Williams  and  Wilkins 
Co.,  Baltimore,  1920. 


THE   PREPARATION   OF  CULTURE    MEDIA  147 

with  tautomeric  rearrangements  of  the  original  substances,  and  the 
state  of  these  substances  in  a  dissociated  or  undissociated  form 
determines  the  color.  The  degree  of  dissociation  as  determined  by 
the  hydrogen  ion  concentration  brings  about  a  predominance  of  one 
or  the  other  tautomeric  compound,  and  the  color  depends  upon 
which  one  of  these  compounds  is  associated  with  the  color.  Chapter 
three  of  Clark's  book  goes  into  this  matter  with  sufficient  thorough- 
ness for  bacteriological  work. 

In  cultural  work  not  many  indicators  are  needed.  For  the  actual 
titration  of  media,  the  necessary  indicators  are  given  in  another 
section.  For  actual  addition  to  culture  media,  we  may  restrict  our- 
selves to  a  limited  number  of  useful  indicators. 

Litmus  is  the  indicator  most  commonly  used  in  former  years,  is 
a  product  obtained  from  a  species  of  lichen,  and  is  obtained  by 
oxidation  of  the  orcin  contained  in  this  plant.  Asolitmin  is  the 
indicator  substance  in  the  litmus,  chemically  complex  and  not  com- 
pletely analyzed. 

The  litmus  solutions  used  in  the  preparation  of  media  are  best 
made  up  as  follows :  Litmus  in  substance — Merck 's  purified,  or  Kaul- 
baum's — is  dissolved  in  water  to  the  extent  of  5  per  cent.  The 
solution  is  made  by  heating  in  an  Arnold  sterilizer  for  about  one 
to  two  hours,  shaking  occasionally.  The  solution  is  then  filtered 
through  paper  and  sterilized.  It  should  be  kept  sterile,  as  molds 
will  grow  in  it  otherwise. 

A  standard  litmus  solution,  which  is  marketed  for  laboratory 
purposes,  known  as  "Kubel  and  Tiemann's"  solution,  may  be  used. 

Andrade  indicator  is  made  up  as  follows: 

0.5  per  cent  aqueous  acid  fuchsin 100  c.c. 

Normal  NaCH    16  c.c. 

The  red  color  fades  out  gradually ;  the  indicator  should  be  yellow 
after  standing  two  or  three  hours.  If  it  remains  red  or  reddish, 
1  c.c.  more  of  normal  NaOH  may  be  added. 

Acid  production  turns  this  indicator  red,  due  to  the  neutralization 
of  the  alkali  present  and  the  liberation  of  the  acid  color  base.  The 
medium  in  which  Andrade  indicator  is  used,  must  be  adjusted  to 
the  neutral  point  of  this  indicator  which  is  equivalent  to  P^  7.2. 
The  reaction  of  the  medium  is  right  for  this  indicator,  when  the 
medium  containing  1  per  cent  of  the  indicator  is  red  when  hot,  and 
colorless  when  cold. 


148  BIOLOGY  AND  TECHNIQTJE 

China,  Blue  Indicator. — Make  up  1  per  cent  solution  of  China  blue, 
heat  almost  to  boiling  in  the  water  bath,  add,  drop  by  drop,  normal 
NaOH  until  completely  decolorized.  Use  1.5  per  cent  concentration 
in  medium.  This  indicator  seems  to  be  much  less  toxic  than  some 
of  the  others,  and  has  been  used  with  success  particularly  by  Teague, 
to  whom  we  are  indebted  for  these  practical  details. 

Neutral  red  an  indicator  formerly  used  extensively  by  the  Ger- 
mans in  the  proportions  of  1  c.c.  of  staturated  aqueous  solution  to 
100  c.c.  of  culture  medium.  It  has  been  particularly  used  in  con- 
nection with  the  colon-typhoid  group,  in  that  typhoid  bacilli  do 
not  change  the  color,  whereas  B.  coli  decolorizes  it. 

The  list  of  indicators  might  be  very  much  increased,  but  there 
would  be  little  point  in  it  since  most  of  these  indicators  are  not 
being  used  at  the  present  time. 

ACTUAL  STEPS  IN  THE  PREPARATION  OP  NUTRIENT 

MEDIA 

Meat  Extract  Broth. — To  1000  c.c,  of  distilled  or  clear  tap  water,  add 
5  grams  or  0.5  per  cent  of  Liebig's  meat  extract,  10  grams  or  1  per  cent 
of  Witte  or  any  other  reliable  brand  of  pepton,  and  5  grams  or  0.5  per  cent 
of  common  salt,  NaCl. 

The  ingredients  are  mixed  together  in  a  suitable  vessel  and  heated  with 
stirring  over  a  free  flame.  When  the  pepton  and  meat  extract  are  completely 
dissolved,  the  vessel  is  removed  from  the  flame.  The  medium  is  titrated 
by  the  colorimetric  method  described  above  and  adjusted  to  the  desired 
reaction.  It  is  advisable  to  make  the  reaction  about  two  points  (on  the 
hydrogen  ion  scale)  more  alkaline  than  the  final  reaction  should  be,  since 
the  heat  of  the  autoclave  usually  increases  the  acidity  of  the  medium.  Using 
Liebig's  meat  extract  and  Digestive  Ferment's  pepton,  we  have  found  that 
the  addition  of  10-12  c.c.  of  normal  NaOH  to  every  liter  of  extract  broth 
usually  brings  the  reaction,  before  autoclaving,  to  7.6,  making  it  about  7.4 
after  autoclaving,  which  is  the  optimum  for  most  pathogenic  bacteria.  After 
the  addition  of  the  alkali,  the  broth  is  autoclaved  for  thirty  minutes  at 
fifteen  pounds  pressure.  The  medium  is  then  filtered,  the  reaction  checked 
and  after  tubing  and  sterilization,  is  ready  for  use.  It  is  not  necessary  in 
most  cases  to  add  eggs  to  extract  broth,  since  it  is  easy  to  obtain  clear 
without  this  step. 

Meat  Infusion  Broth. — Infuse  500  grams8  of  lean  meat  (veal  or  beef) 
in  1000  c.c.  of  distilled  or  tap  water  for  twelve  to  twenty -four  hours  in  the 
ice-box.  Strain  through  wet  cheese  cloth,  squeezing  the  meat  as  dry  as 

"Koughly  1  pound  (1*4  Ibs.). 


THE   PREPARATION  OF  CULTURE   MEDIA  149 

possible.  Make  up  the  volume  to  1000  e.c.  Add  1  per  cent  pepton  and 
0.5  per  cent  salt.  Heat  over  the  free  flame  until  the  pepton  is  dissolved, 
stirring  from  time  to  time.  Solution  usually  takes  place  so  rapidly  that 
the  loss  of  water  through  evaporation  is  negligible.  If  desired,  however, 
the  medium  can  be  measured  after  this  preliminary  heating,  and  the  loss 
in  volume  made  up  by  the  addition  of  water.  Titrate  the  medium  and  bring 
it  to  the  desired  reaction  using  the  colorimetric  method.  Meat  infusion  media 
are  usually  considerably  more  acid  than  meat  extract  media.  The  addition 
of  about  20  e.c.  normal  NaOH  per  liter  in  the  case  of  meat  infusion  broth 
usually  brings  the  reaction  to  about  7.4  after  autoclaving.  The  change  in 
reaction  after  autoclaving  is  sometimes  considerable,  and  must  be  carefully 
checked.  After  the  addition  of  the  alkali,  the  medium  is  autoclaved  for 
thirty  minutes  at  fifteen  pounds  pressure.  The  broth  is  now  filtered  through 
paper  or  cotton,  and  usually  comes  through  clear  without  any  further  trouble. 
After  tubing  and  sterilization,  the  medium  is  ready  for  use. 

"Hormone  Medium." 9 — A  curious  observation  has  been  made  recently 
which  seems  to  indicate  that  the  filtration  of  media  removes  from  them 
certain  substances  which  considerably  enhance  their  nutritive  value  for 
bacteria. 

These  substances,  for  want  of  a  better  name,  have  been  spoken  of  as 
hormones,  and  the  hormone  media,  which  are  used  pretty  generally,  and 
which  we  have  found  to  possess  unusual  advantages  over  ordinary  culture 
media,  and  which,  consequently,  we  are  using  almost  altogether  in  our  routine 
work,  are  made  as  follows,  the  description  given  being  that  of  Huntoon : 10 

The  basis  for  "hormone  media"  is  beef  heart  instead  of  the  customary 
beef  or  veal.  It  is  important  that  the  hearts  are  fairly  fresh.  The  heart 
muscle  is  cut  up  in  the  usual  way  ai  d  after  the  removal  of  fat  and  large 
vessels,  put  through  the  meat  grinder.  The  chopped  meat  is  weighed  and 
1  liter  of  water  added  for  every  500  grams  of  meat.  One  per  cent  pepton 
and  0.5  per  cent  salt  are  added  directly,  and  1  egg  (whole)  added  for  each 
liter  of  medium.  If  the  bouillon  is  to  be  used  for  broth,  1  per  cent  gelatin 
is  added  immediately.  If  the  bouillon  is  to  be  used  as  a  basis  for  agar, 
it  is  not  necessary  to  add  the  gelatin.  The  3  per  cent  agar,  finely  cut  up, 
is  added  to  the  other  ingredients. 

When  all  the  ingredients  have  been  placed  in  the  same  pot,  the  mixture 
is  heated  over  the  free  flame  until  it  reaches  a  temperature  of  about  70°  C. 
and  meat  begins  to  turn  brown.  25  e.c.  of  normal  NaOH  are  then  added 
per  liter.  The  pot  is  placed  in  the  Arnold  and  allowed  to  cook  for  1^ 
to  2  hours.  At  the  end  of  this  time,  a  firm  clot  has  usually  formed  and 
the  broth  or  agar  can  be  decanted. 


9  Cole  and  Lloyd,  Journal  Path,  and  Baet.  vol.  21,  1916. 

10  Huntoon,  F.  M.,  Jour,  of  Inf.  Dis.,  23,  1918,  169. 


150  BIOLOGY  AND  TECHNIQUE 

The  medium  is  then  titrated  accurately  and  brought  to  the  desired  reaction. 
The  medium  usually  gets  slightly  more  acid  on  autoclaving,  so  that  it  is 
better  to  adjust  to  about  2  points  more  alkaline  on  the  hydrogen  ion  scale, 
than  the  final  reaction  desired.  It  is  important,  in  making  hormone  media, 
never  to  filter  in  any  way.  Cotton,  paper,  and  cheese  cloth  filters  are  equally 
undesirable.  If  a  firm  clot  is  obtained  after  the  first  heating,  there  is  usually 
no  difficulty  in  obtaining  a  clear  medium.  •  On  the  first  autoclaving  a  second 
precipitate  usually  forms,  from  which  the  clear  medium  can  easily  be  decanted. 
It  is  best  to  check  up  the  reaction  after  the  final  sterilization.  The  Hormone 
media  can  also  be  cleared  with  the  Sharpless  centrifuge. 

Sugar-free  Broth. — Make  one  liter  of  infusion  broth11  according  to  the 
directions  given.  Inoculate  with  a  young  culture  of  B.  coli  comnmnis. 
Incubate  for  twenty-four  to  forty-eight  hours.  The  bacteria  will  ferment 
and  thus  destroy  any  sugar  (mono-saccharide)  which  may  be  present  in  the 
broth,  and  thus  render  the  medium  sugar-free  and  acid. 

Arnold  for  one  hour  to  kill  the  B.  coli. 

Titrate  and  adjust. 

Arnold  again  for  thirty  minutes. 

Filter  through  paper  until  clear. 

This  sugar-free  medium  is  used  as  a  basis  for  fermentation  reactions.  The 
different  sugars  are  added  in  1  per  cent  concentrations  and  the  medium  is 
then  sterilized  for  three  successive  days  in  the  Arnold,  since  the  higher 
temperatures  of  the  autoclave  tend  to  split  the  more  complex  sugars  into 
the  simpler  ones. 

Glycerin  Broth. — To  ordinary,  slightly  acid  or  neutral  meat  infusion 
broth,  add  six  per  cent  of  C.  P.  glycerin.  Sterilize  by  fractional  method. 

Calcium  Carbonate  Broth. — This  medium  is  designed  for  obtaining  mass 
cultures  of  pneumococcus  or  streptococcus  for  purposes  of  immunization  or 
agglutination. 

To  100  c.c.  of  meat  infusion  broth  in  small  flasks,  add  one  per  cent 
of  powdered  calcium  carbonate,  and  one  per  cent  of  glucose.  It  is  a  wise 
precaution  to  sterilize  the  dried  calcium  carbonate  in  the  hot-air  chamber 
before  using.  Small  pieces  of  marble  may  be  used  as  suggested  by  Bolduan. 

METHOD  FOR  THE  PREPARATION  OF  NUTRIENT  BROTH12  (Avery)  (for 
Streptococcus  Agglutination). — One  pound  of  lean  chopped  beef  allowed  to 
infuse  in  a  liter  of  tap  water  over  night  in  the  ice-box.  The  unfiltered  meat 
infusion  boiled  for  30  minutes,  filtered  through  paper,  and  the  loss  by  evapora- 
tion made  up  by  the  addition  of  water.  One  per  'cent  peptone  (Faircliild) 
and  0.2  per  cent  sodium  phosphate  (Na.JIPOJ  are  now  added.  The  mixture 


"It  is  not  necessary  to  filter  the  infusion  if  it  is  to  be  used  for  making 
sugar  free  broth. 

12  We  may  add  that  this  method  of  growing  streptococcus  for  agglutination 
purposes  does  not  always  work  out  successfully — but  is  the  best  we  know  of  so  far. 


THE   PREPARATION    OF   CULTURE   MEDIA  151 

is  allowed  to  boil  for  20  minutes  and  the  reaction  is  adjusted  to  the  desired 
P7/  (About  0.2  of  a  PH  is  allowed  for  change  in  the  reaction  during 
sterilization.  For  pneumococcus  work  the  optimus  P//  is  7.8;  in  the  adjust- 
ment, therefore,  before  sterilization,  the  reaction  is  set  at  PH  8.)  The  broth 
is  sterilized  in  the  Arnold  sterilizer  for  20  minutes  on  three  successive  days. 
For  streptococcus  work  the  final  read  ion  should  be  P//  7.4. 
Pepton-Salt  Solution  (Dunham's  solution)  : 

1.  Distilled   water    1,000    c.c. 

Pepton  (Witte)   10  gms. 

NaCl    5     < ' 

2.  Heat  until  ingredients  are  thoroughly  dissolved. 

3.  Filter  through  filter  paper  until  perfectly  clear. 

4.  Tube  or  store  flasks. 
Sterilize  by  discontinuous  method. 
Nitrate  Solution: 

1.  Distilled  water    1,000       c.c. 

Pepton 10      gms. 

Potassium  nitrate   10      gms. 

2.  Heat  until  ingredients  are  thoroughly  dissolved. 

3.  Filter  through  filter  paper  until  perfectly  clear. 

4.  Tube  or  store  flasks. 

Sterilize  by  discontinuous  sterilization. 

Uschinsky's  Protein-Free  Medium.13 — To  one  liter  of  distilled  water  add: 

Asparagin    3.4  grams. 

Ammonium  lactate  10  * ' 

Sodium  chlorid    5  ' ' 

Magnesium   sulphate    0.2  ' ' 

Calcium  chlorid 0.1  • ' 

Potassium  phosphate   1.0  ' ' 

When  these  substances  are  thoroughly  dissolved,  add  40  c.c.  of  glycerin.  Tube 
and  sterilize. 

Meat  Extract  Gelatin. — Gelatin  is  best  made  in  the  Arnold  and  should 
at  no  stage  be  autoclaved. 

One  per  cent  pepton,  0.5  per  cent  salt,  0.5  per  cent  meat  extract  in  one 
liter  of  water  are  dissolved  in  the  Arnold.  When  these  ingredients  have 
been  dissolved,  15  to  18  per  cent  of  finest  French  sheet  gelatin14  are  added, 
and  the  mixture  kept  in  the  Arnold  until  the  gelatin  is  completely  dissolved. 

13  Uschinsky,  Cent.  f.  Bakt.,  1,  xiv,  1893. 

14  The  acidity  and  consistence  of  the  different  commercial  gelatins  vary  con- 
siderably and   care  should  be  taken  in  selecting  a  uniform  and  suitable  brand, 
such  as  Hesterberg  '&  gold  label  gelatin.     It  is  advisable,  when  working  during 
the  summer  or  in  hot  climates,  to  add  18  per  cent. 


152  BIOLOGY  AND  TECHNIQUE 

The  medium  is  then  titrated  and  adjusted.  After  cooling,  the  whites  of 
two  eggs  are  added  and  the  media  is  put  in  the  Arnold  for  forty-five  minutes. 
The  medium  is  filtered,  tubed  and  sterilized  by  the  fractional  method.  All 
unnecessary  heating  of  gelatin  is  to  be  avoided. 

Meat  Infusion  Gelatin. — Meat  infusion  gelatin  is  made  in  the  same  way 
as  the  above  except  that  fresh  meat  infusion  is  substituted  for  the  meat 
extract. 

Meat-Extract  Agar. — To  1  liter  of  distilled  or  tap  water  add: 

Thread  agar   15  to  30.0  grams15 

Pepton   10.0       ' ' 

. .     Meat  extract .     5.0       " 

Common  salt 5.0       " 

Put  in  autoclave,  15  pounds'  pressure,  for  15  minutes.  The  agar  can 
also  be  dissolved  over  the  free  flame,  but  this  takes  a  long  time  and  is  not 
necessary.  However,  if  so  done  make  up  loss  by  evaporation. 

Take  out  of  autoclave,  and  adjust  to  desired  reaction. 

Cool  to  60°  C.  and  add  the  whites  of  two  eggs.     Stir  thoroughly. 

Heat  either  in  the  autoclave,  fifteen  pounds,  thirty  minutes;  or  if  no 
autoclave  is  available,  thirty  minutes  in  the  Arnold.  If  the  heating  is  done 
in  the  Arnold,  take  out  after  half  an  hour,  stir  and  replace  for  fifteen 
minutes  more. 

Titrate,  and  adjust  again  if  reaction  has  changed. 

If  a  correction  in  reaction  is  made,  heat  again  for  ten  minutes,  filter 
through  cotton,  tube  and  sterilize. 

Meat  Infusion  Agar.16 — In  making  meat  infusion  agar  it  is  best  to  prepare 
two  solutions,  one  consisting  of  the  meat  infusion  to  which  the  pepton  and 
salt  are  added,  the  other  an  aqueous  solution  of  agar  which  is  dissolved 
in  the  autoclave.  The  ingredients  are  added  in  double  strength  to  each 
solution  so  that  when  they  are  finally  combined,  the  desired  concentrations 
are  obtained. 

Solution  (1)  500  grams  of  lean  meat  in  500  c.c.  of  distilled  or  tap  water 
are  infused  over  night  in  the  ice-box.  The  infusion  is  then  strained  through 
cheese  cloth  and  all  the  juice  squeezed  out  of  the  meat.  The  infusion  is 
measured  and  the  volume  made  up  to  500  c.c.  Two  per  cent  pepton  or 
10  grams,  and  1  per  cent  or  5  grams  salt  are  now  added  to  the  500  c.c. 
infusion  and  the  mixture  is  heated  over  ihe  free  flame  until  it  reaches  a 
temperature  of  about  50°  C.  and  the  pepton  dissolves.  This  solution  is  then 


15  Amount  of  agar  varies  according  to  stiffness  desired. 

16  While  titrating  care  should  be  taken  that  the  medium  does  not  solidify  along 
the  sides  of  the  vessel.     Glycerin  agar  is  made  by  adding  6  per  cent  C.P.  glycerin 
to  meat  extract  or  meat  infusion  agar. 


THE  PREPARATION  OF  CULTURE   MEDIA  153 

titrated  and  sufficient  alkali  added  to  make  the  reaction  about  two  or  three 
points  more  alkaline  than  the  desired  final  reaction  of  the  medium. 

Solution  (2)  to  500  c.c.  of  water  add  the  quantity  of  agar  that  will 
give  the  right  concentration  for  the  final  volume  (1  liter).  For  instance, 
if  a  3  per  cent  agar  is  desired,  add  30  grams  of  agar  to  500  c.c.  of  water 
and  dissolve  in  the  autoclave  at  fifteen  pounds  pressure  for  fifteen  minutes. 
When  the  agar  is  dissolved,  cool  it  down  to  50°  C.  and  add  to  it  Solution  (1). 
Mix  thoroughly  and  titrate  again.  Usually  the  addition  of  the  agar  solution 
does  not  change  the  reaction.  Add  the  whites  of  two  eggs  well  beaten  with 
a  little  water  and  autoclave  for  thirty  minutes  at  fifteen  pounds  pressure. 
If  no  autoclave  is  available,  the  medium  may  be  put  in  the  Arnold  for 
forty-five  minutes.  The  autoclave,  however,  is  more  satisfactory.  It  is  best 
to  check  the  titration  again  after  autoclaving. 

The  medium  is  now  filtered,  tubed  and  sterilized. 

LACTOSE-LITMUS- AGAR  (Wurtz). — This  is  ordinary  meat  extract  agar 
which  is  adjusted  to  P^  7.5  to  7.8,  to  which  1  per  cent  of  lactose  is  added, 
and  enough  litmus  to  give  it  a  bluish  purple  color  when  cooled.  Instead 
of  litmus,  1  per  cent  of  the  Andrade  indicator  can  be  used.  If  the  latter 
indicator  is  used,  the  reaction  of  the  medium  must  be  brought  down  to  P^ 
7.2.  The  medium  containing  1  per  cent  of  the  indicator,  if  the  reaction  is 
correct,  will  be  dark  red  when  hot  and  colorless  when  cold.  After  the 
addition  of  the  sugar  and  the  indicator,  it  is  best  to  sterilize  by  the  inter- 
mittent method  on  three  successive  days. 

The  red  color  fades  out  gradually,  the  indicator  should  be  yellow  after 
standing  2  or  3  hours.  If  it  remains  red  or  reddish,  1  or  2  c.c.  more  of 
normal  NaOH  should  be  added. 

The  above  description  of  the  basic  media,  broth  and  agar,  details  chiefly 
the  methods  in  general  use  until  a  few  years  ago.  The  recognition  that 
bacteria  grow  more  luxuriantly  upon  media  containing  considerable  quantities 
of  protein-split  products,  has  resulted  in  the  utilization  of  trypsinized  culture 
media  which  has  been  found  to  possess  considerable  advantages  for  the 
cultivation  of  delicate  organisms  like  the  meningococci,  etc.  A  simple  formula 
for  the  production  of  trypsinized  agar  is  the  following,  taken  from  the 
directions  of  Gordon  :  17 

TRYPAGAR. — To  one  pound  of  chopped  meat,  free  from  fat,  add  1  liter 
tap  water  and  make  faintly  alkalin  to  litmus  with  20  per  cent  NaOH 
solution.  Heat  in  double  boiler  at  75°  to  80°  for  five  minutes.  Cool  to  37° 
and  add  0.5  gram  trypsin.  (Fairchild's  preparation.  For  other  brands  the 
amount  must  be  determined  by  experiment.)  Incubate  for  five  to  six  hours. 
Test  for  pepton  as  follows:  Take  5  c.c.  of  the  liquid,  add  5  c.c.  N/l  NaOH 
and  1  c.c.  dilute  CuSO4.  A  pink  color  indicates  that  trypsinization  is 


17  Gordon,  Br.  Med.  Journal  2,  1916,  678. 


154  BIOLOGY  AND  TECHNIQUE 

complete — a  bluish  purple  shade,  that  it  is  incomplete.  If  test  is  satisfactory, 
slightly  acidify  the  broth  with  glacial  acetic  acid  and  bring  slowly  to  boiling 
point  and  boil  gently  for  ten  minutes  and  filter  through  paper.  Add  2  per 
cent  agar  and  0.5  per  cent  salt,  autoclave  to  dissolve  agar,  and  proceed  from 
this  point  as  usual,  clearing  with  egg  and  setting  to  P  H  7.5,  or  any  desired 
reaction. 

Vedder  Starch  Agar. — Beef  infusion  agar  is  prepared  without  salt  and 
pepton.  This  is  adjusted  to  P^  6.8,  10  grams  of  corn  starch  is  added  to 
each  liter,  and  the  mixture  is  heated  in  the  autoclave  for  20  minutes  at  15 
pounds.  It  is  tubed  and  sterilized.  This  medium  has  been  recommended  by 
Vedder  for  the  cultivation  of  gooococcus. 

Welch's  Modification  of  Guarnieri's  Medium.™ — This  medium  is  made  on 
a  meat  infusion  basis,  according  to  the  directions  given  for  the  preparation 
of  meat-infusion  agar.  It  contains  5  grams  of  agar,  80  grams  of  gelatin, 
5  grams  of  NaCl,  and  10  grams  of  pepton  to  one  liter.  It  should  be  adjusted 
to  a  neutral  reaction.  It  is  used  for  stab  cultures  and  is  designed  chiefly 
for  pneumococcus  cultivation  and  storage. 

Dorsett  Egg  Medium. — This  medium  is  chiefly  useful  for  the  cultivation 
of  tubercle  bacilli. 

1.  Carefully  break  eggs  and  drop  the  contents  into  a  wide-mouthed  flask. 
Break  up  the  yolk  with  a  sterile  platinum  wire,  and  shake  up  the  flask  until 
the  whites  and  yolks  are  thoroughly  mixed. 

2.  Add  25  c.c.  of  distilled  water  to  every  four  eggs;  strain  through  sterile 
cloth. 

3.  Pour  10  c.c.  each  into  sterile  test  tubes  and  slant  in  an  inspissator 
and  expose  to  73°  C.  for  four  to  five  hours  on  two  days. 

4.  On  the  third  day,  raise  the  temperature  to  76°  C. 

5.  The  sterilization  may  be  finished  by  a  single  exposure  to  100°   C.  in 
the  Arnold  sterilizer  for  fifteen  minutes.     Before  inoculation,  add  two  or 
three  drops  of  sterile  water  to  each  tube. 

LUBENAU'S  GLYCERIN- EGG. — To  1  liter  of  veal  broth  containing  2  per 
cent  pepton,  5  per  cent  of  glycerin  is  added.  Neutralize  this  to  litmus, 
and  to  every  200  c.c.  add  10  fresh  eggs.  The  mixture  is  thoroughly  stirred, 
and  when  homogeneous,  is  tubed,  slanted  and  inspissated,  as  in  the  case  of 
other  egg  media. 

PETROFIF'S  MEDIUM.— I.  Meat  Juice.  500  grams  of  beef  or  veal  are 
infused  in  500  c.c.  of  a  15  per  cent  solution  of  glycerin  in  water,  in  a  cool 
place.  After  24  hours  the  meat  is  squeezed  in  a  sterile  press  and  the  infusion 
collected  in  a  sterile  beaker. 

II.  Eggs.  The  shells  of  the  eggs  are  sterilized  by  10  minute  immersion 
in  70  per  cent  alcohol.  They  are  broken  into  a  sterile  beaker,  well  mixed 


"  Welch,  Bull.  Johns  Hopkins  Hosp.,  1892,  vol.  3,  p.  127. 


THE  PREPARATION  OF  CULTURE   MEDIA  155 

and  filtered  through  sterile  gauze.  One  part  of  meat  juice  is  added  to  two 
parts  of  egg  by  volume. 

III.  Gentian  Violet.  1  per  cent  alcoholic  solution  of  gentian  voilet  is 
added  to  make  a  final  proportion  of  1  :  10,000. 

The  three  ingredients  are  well  mixed.  The  medium  is  tubed  and  inspis- 
sated as  usual. 

Petroff  recommends  for  sputum  the  following  technique:  Equal  parts 
of  sputum  and  3  per  cent  sodium  hydroxid  are  shaken  and  incubated  at 
38°  C.  for  15  to  30  minutes,  the  time  depending  on  the  consistency  of  the 
sputum.  The  mixture  is  neutralized  to  litmus  with  hydrochloric  acid  and 
centrifugalized.  The  sediment  is  inoculated  into  the  medium  described  above. 
Pure  cultures  are  obtained  in  a  large  proportion  of  cases. 

SYNTHETIC  MEDIA  FOR  THE  TUBERCLE  BACILLUS. — A  considerable  number 
of  synthetic  media  have  been  made  for  the  growth  of  the  tubercle  bacillus. 
The  purpose  of  these  media  is  to  omit  complex  protein  substances  as  much 
as  possible.  The  one-  we  give  below  is  according  to  the  formula  used  by 
Petroff  with  success. 

0.35  gram K2HPO4 

4.93  grams Mg  HPO4.  2H2O 

10  c.c normal  H2SO4 

20  c.c normal   (1/3  molar)   H3PO4 

10  c.c 3  times  normal    (molar)     citric  acid 

5.29  grams asparagin 

20  c.c glycerin 

1000  c.c water 

add  10  c.c.  normal  NaOH. 

Potato  Media. — Large  potatoes  are  selected,  washed  in  hot  water,  and 
scrubbed  with  a  brush.  They  are  peeled,  considerably  more  than  the  cuticle 
being  removed.  The  peeled  potatoes  are  washed  in  running  water,  following 
which  cylindrical  pieces  are  removed  with  a  large  apple  corer.  The  cylinders 
are  cut  into  wedges. 

Since  the  reaction  of  the  potato  is  normally  acid,  this  should  be  corrected 
by  washing  the  pieces  in  running  water  over  night,  or,  better,  by  immersing 
them  in  a  one  per  cent  solution  of  sodium  carbonate  for  half  an  hour. 

The  pieces  are  then  inserted  into  the  large  variety  of  test  tubes  known 
as  "potato  tubes."  (See  Fig.  21,  c.)  In  the  bottom  of  the  tubes  a  small 
amount  of  water  (about  1  c.c.)  or  a  small  quantity  of  moist  absorbent  cotton 
should  be  placed  in  order  to  retard  drying  out  of  the  potato.  The  tubes 
are  sterilized  by  fractional  sterilization,  twenty  minutes  to  half  an  hour  in 
the  Arnold  sterilizer  on  three  successive  days. 

POTATO  BROTH. — Petroff  has  used  extract  of  potatoes  in  fluid  media  for 
the  growth  of  tubercle  bacilli.  There  are  many  different  ways  of  preparing 
the  potato  extract.  The  best  way  is  to  finely  grind  thoroughly,  or  grate  the 


156  BIOLOGY  AND  TECHNIQUE 

potatoes  and  soak  them  in  tap  water  for  from  12  to  24  hours,  using  about 
500  grams  to  a  liter  of  water.  This  mixture  can  be  filtered,  or,  better, 
heated  before  filtration.  It  may  be  used  as  an  ingredient  with  or  without 
glycerin  in  ordinary  broth  or  agar,  or  can  be  used  with  pepton  and  salt 
added,  as  an  independent  culture  medium. 

Glycerin  Potato. — In  preparing  glycerin  potato  the  potato  wedges  are 
treated  as  above,  and  are  then  soaked  in  a  ten  to  twenty-five  per  cent  aqueous 
glycerin  solution  for  one  to  three  hours.  A  small  quantity  of  a  ten  per 
cent  glycerin  solution  should  be  left  in  the  tubes.  In  sterilizing  these  tubes, 
thirty  minutes  a  day  in  the  Arnold  after  the  sterilizer  is  hot,  will  sterilize 
without  altering  the  glycerin. 

Milk  Media. — Fresh  milk  is  procured  and  is  heated  in  a  flask  for  fifteen 
minutes  in  an  Arnold  sterilizer.  It  is  then  set  away  in  the  ice  chest  for 
about  twelve  hours  in  order  to  allow  the  cream  to  rise.  Milk  and  cream 
are  then  separated  by  siphoning  the  milk  into  another  flask.  It  is  rarely 
necessary  to  adjust  the  reaction  of  milk  prepared  in  this  way,  since,  if  acid 
at  all,  it  is  usually  but  slightly  so.  If,  however,  it  should  prove  more  than 
1.5  per  cent  acid,  it  should  be  discarded  or  neutralized  with  sodium  hydrate. 
The  milk  may  then  be  tubed  either  with  or  without  the  addition  of  an 
indicator.  Litmus  gives  the  most  satisfactory  results  in  milk,  but  at  the 
present  time  is  difficult  to  obtain.  The  Andrade  indicator  can  be  used  also, 
but  usually  discolors  the  milk  somewhat.  However,  if  fractional  sterilization 
is  carefully  carried  out,  the  milk  becomes  yellowish,  but  acid  production 
shows  up  clearly  by  a  distinct  reddening  of  the  medium.  If  the  milk  is 
to  be  used  for  the  differentiation  of  the  anaerobic  bacilli  isolated  from  war 
wounds,  it  is  best  not  to  add  any  indicator,  since  the  type  of  coagulum 
formed  is  a  differential  characteristic,  and  coagulation  does  not  take  place 
readily  when  Andrade  is  present. 

SODIUM  OLEATE  AGAR  (For  Influenza  Bacilli). — Avery19  has  found  that 
sodium  oleate  will  enhance  the  development  of  influenza  bacilli,  and  at  the 
same  time  will  inhibit  many  of  the  Gram-positive  organisms  commonly  found 
in  sputum. 

A  neutral  solution  of  Kahlbaum's  sodium  oleate  in  water  is  prepared  and 
sterilized  in  the  autoclave.  Human  or  rabbit  blood  is  defibrinated,  centri- 
fuged,  the  serum  removed,  and  the  volume  made  up  to  the  original  with 
broth.  1  c.c.  of  the  red  blood  cell  suspension  and  5  c.c.  of  the  2  per  cent 
sodium  oleate  solution  are  added  to  94  c.c.  of  agar  at  80°  to  90°  C.  The 
agar  is  preferably  a  2  per  cent  hormone  agar  with  a  reatcion  of  Prt  7.4. 

Serum  Media. — Loeffler's  Medium. — Beef  blood  is  collected  at  the 
slaughter  house  in  high  cylindrical  jars  holding  two  quarts  or  more.  It  is 
desirable  that  attempts  should  be  made  to  avoid  contamination  as  much  as 


19  J.  A.  M.  A.  1918,  vol.  71,  2050. 


THE   PREPARATION   OF  CULTURE   MEDIA  157 

is  feasible  by  previously  sterilizing  the  jars,  keeping  them  covered,  and 
exercising  care  in  the  collection  of  the  blood. 

The  blood  is  allowed  to  coagulate  in  the  jars,  and  should  not  be  moved 
from  the  slaughter  house  until  coagulated.  All  unnecessary  shaking  of  jars 
should  be  avoided.  As  soon  as  the  coagulum  is  fully  formed,  adhesions 
between  the  clot  and  the  sides  of  the  jar  should  be  carefully  separated  with 
a  sterile  glass  rod  or  wire.  The  jars  are  then  set  away  in  the  ice  chest  for 
24  to  36  hours.  At  the  end  of  this  time  clear  serum  will  be  found  over 
the  top  of  the  clot,  and  between  the  clot  and  the  jar.  This  should  be  pipetted 
off,  preferably  with  a  large  pipette  of  50  to  100  c.c.  capacity,  or  siphoned 
off  with  sterile  glass  tubing,  and  transferred  to  sterile  flasks. 

To  three  parts  of  the  clear  serum  is  then  added  one  part  of  a  one  per 
cent  glucose  beef  infusion  or  veal  infusion  bouillon.  The  mixture  is  filled 
into  tubes,  preferably  the  short  test  tubes  commonly  used  for  diagnostic 
diphtheria  cultures.  The  tubes  are  then  placed  in  a  slanting  position  in 
the  apparatus  known  as  an  inspissator  (see  p.  71).  This  is  a  double- 
walled  copper  box  covered  by  a  glass  lid,  eased  in  asbestos,  and  •surrounded 
by  a  water  jacket.  It  is  heated  below  by  a  Bunsen  flame.  Together  with 
the  tubes  a  small  open  vessel  containing  water  should  be  placed  in  the" 
inspissator  to  insure  sufficient  moisture.  The  temperature  of  the  inspissator 
is  now  raised  to  70° -75°  C.,  care  being  taken  that  the  rise  of  temperature 
takes  place  slowly.  The  temperature  is  maintained  at  this  point  for  two 
hours,  and  the  process  is  repeated,  for  the  same  length  of  time,  at  the  same 
temperature,  on  six  successive  days,  preferably  without  removing  the  tubes 
from  the  inspissator  at  any  time.  It  is  also  possible,  though  less  regularly 
yielding  good  results,  to  sterilize  in  the  inspissator  for  one  day,  following 
this  on  the  second  and  third  days  by  exposure  for  thirty  minutes  to  100°  C. 
in  the  Arnold  steam  sterilizer.  In  doing  this,  the  Arnold  should  be  very 
gradually  heated,  at  first  without  outer  jacket,  this  being  lowered  only  after 
thorough  heating  has  taken  place. 

Serum-Water  Media  for  Fermentation  Tests. — For  the  determination  of 
the  fermentative  powers  of  various  microorganisms  for  purposes  of  differen- 
tiation, Hiss  has  devised  the  following  media  in  which  the  cleavage  of  any 
given  carbohydrate  is  indicated,  not  only  by  the  production  of  an  acid 
reaction,  but  by  the  coagulation  of  the  serum  proteins. 

Obtain  clear  beef  serum  by  pipetting  from  clotted  blood  in  the  same 
way  as  this  is  obtained  for  the  preparation  of  Loeffler's  blood-serum  medium. 
Add  to  this  two  or  three  times  its  bulk  of  distilled  water,  making  a  mixture 
of  serum  and  water  in  proportions  of  one  to  two  or  three.  Heat  the 
mixture  for  fifteen  minutes  in  an  Arnold  sterilizer  at  100°  C.  to  destroy 
any  diastatic  ferments  present  in  the  serum.  Add  one  per  cent  of  a  five 
per  cent  aqueous  litmus  solution  (the  variation  in  the  different  litmus 
preparations  as  obtained  in  laboratories  necessitates  a  careful  addition  of 


158  BIOLOGY  AND  TECHNIQUE 

an  aqueous  litmus  solution  until  the  proper  color,  a  deep  transparent  blue, 
is  obtained,  rather  than  rigid  adherence  to  any  quantitative  directions).  With 
many  batches  of  serum,  it  will  be  found  that  the  addition  of  two  or  three 
times  its  bulk  of  distilled  water  is  not  a  sufficient  dilution  to  prevent 
coagulation.  It  will  often  be  found  necessary  to  add  four  or  five  volumes 
of  distilled  water  to  one  volume  of  serum.  One  per  cent  of  the  Andrade 
indicator  may  be  substituted  for  the  litmus.  Add  to  the  various  fractions 
of  the  medium  thus  made  one  per  cent  respectively  of  the  sugars  which  are 
to  be  used  for  the  tests. 

For  the  preparation  of  inulin  medium,  made  in  this  way  for  pneumococcus- 
streptococcus  differentiation,  it  is  necessary  to  sterilize  the  inulin  dissolved 
in  the  water  to  be  added  to  the  serum  in  an  autoclave  at  high  temperature 
(15  pounds  for  15  minutes)  in  order  to  kill  spores  before  mixing-  with  the 
•  serum.  The  serum-water  media  are  sterilized  by  the  fractional  method  at 
100°  C.,  at  which  temperature  they  remain  fluid. 

Chocolate  Media  (Park  and  Williams). — It  has  recently  been  observed 
that  for  the -cultivation  of  organisms  like  the  influenza  bacillus,  meningococcus, 
and  a  number  of  other  of  the  more  delicately  growing  bacteria,  an  excellent 
medium  can  be  made  up  in  the  following  way:  Agar  or  broth  are  made 
up  as  usual,  and  to  them  added  defibrinated  rabbit,  beef,  horse  or  human  blood 
in  proportions  of  from  5  per  cent  to  .10  per  cent  by  volume.  This  mixture 
is  then  heated  gradually  up  to  about  75°,  until  the  blood  begins  to  coagulate 
and  assume  a  dark  brown  chocolate  like  color.  The  broth  or  agar  can  first 
be  adjusted  to  the  desired  reaction,  but  it  is  likely  that  any  excess  alkali 
or  acidity  is  corrected  by  the  proteins  which  are  added.  The  medium  can 
be  tubed,  or,  in  the  case  of  agar,  plated  or  slanted,  as  it  is,  after  distribution 
of  the  blood  throughout  the  medium  by  shaking. 

In  the  case  of  broth  the  medium  can  be  filtered  through  paper  while 
hot,  and  sterilized  subsequently  by  filtration  and  fractional  heating.  Such 
filtrate  consists  of  a  clear  brownish  fluid  on  which  influenza  bacilli  and  other 
organisms  grow  with  enormous  speed.  The  speed  with  which  influenza  bacilli 
grow  on  this  medium,  and  its  almost  complete  freedom  of  hemoglobin,  but 
very  much  increased  cholestrin  and  other  lipoid  contents,  have  lead  us  to 
believe  that  it  is  not  the  hemoglobin  particularly  which  is  needed  for  influenza 
bacillus  cultivation. 

SPECIAL  MEDIA  FOR  COLON  TYPHOID  DIFFERENTIATION 

Conradi-Drigalski  Medium.-®— Original  directions. 

(a)  Three  pounds  of  meat  are  infused  in  two  liters  of  water  for  twelve 
hours  or  more.  Strain,  boil  for  one  hour  and  add  20  gms.  Witte's  pepton, 
20  gms.  of  nutrose,  10  gins,  of  NaCl ;  boil  one  hour  and  filter.  Add  60  gms. 


(Jonrvdi- VripateM,  Zeit,  f,  Hyg.z  xxxix,  1902, 


THE   PREPARATION   OF  CULTURE   MEDIA  159 

of  agar.  Boil  for  three  hours  (or  one  hour  in  an  autoclave)  until  agar  is 
dissolved.  Render  weakly  alkaline  to  litmus  paper,  filter,  and  boil  for  half 
an  hour  more. 

(b)  Litmus  solution:  Two  hundred  and  sixty  e.c.  of  litmus  solution  are 
boiled  for  ten  minutes.     (The  litmus  solution  used  by  Conradi  and  Drigalski 
is  the  very  sensitive  aqueous  litmus  recommended  by  Kubel  and  Tiemann, 
and  purchasable  under  the  name.)     After  boiling,  30  grams  of  chemically 
pure  lactose  are  added  to  the  litmus  solution.     The  mixture  is  then  boiled 
for  fifteen  minutes,  and  if,  a  sediment  has  formed,  is  carefully  decanted. 

(c)  Add  to  the  hot  lactose  mixture  to  the  hot  agar  solution;  mix  well 
and,  if  necessary,  again  adjust  to  weak  alkaline  reaction,  litmus  paper  being 
used  as  an  indicator.     To  this  mixture  add  4  c.c.  of  a  hot,  sterile  ten  per  cent 
solution  of  sodium  carbonate,   and  20   c.c.   of  a  freshly   made   solution  of 
crystal  violet  (c.  p.  Hochst),  0.1  gram  in  100  c.c,  of  sterile  distilled  water. 

Surface  smears  are  made  upon  large  plates.  These  are  incubated 
twenty-four  hours.  Typhoid  colonies  are  small,  blue,  and  trans- 
parent. Colon  colonies  are  large,  red,  and  opaque. 

Endo's  Medium.-1 — 1.  Prepare  one  liter  of  meat  infusion  three  per  cent 
agar,  containing  10  grams  of  pepton  and  5  grams  of  NaCl. 

2.  Neutralize  and  clear  by  filtration. 

3.  Add  10  c.c.  of  10%  sodium  carbonate  to  render  alkaline. 

4.  Add  10  grams  of  chemically  pure  lactose. 

5.  Add  5  c.c.  of  alcoholic  fuchsin  solution,  filtered  before  using.     Endo 
in  his  original  contribution  does  not  mention  the  strength  of  this  fuchsin 
solution,  which,  however,  should  be  saturated.     This  colors  the  medium  red. 

6.  Add  25  c.c.  of  a  10%  sodium  sulphite  solution.     This  again  decolorizes 
the  medium,  the  color  not  entirely  disappearing,  however,  until  the  agar  is 
cooled. 

7.  Put  into  test  tubes,  15  c.c.  each,  and  sterilize. 

The  medium  should  be  kept  in  dark.  Plates  are  poured  and  surface 
smears  made.  The  typhoid  colonies  remain  colorless,  while  those  of  coli 
become  red. 

The  preparation  of  Endo's  medium  presents  difficulties  due  to 
the  varying  purity  of  sodium  sulphite.  Kastle  and  Elvove22  recom- 
mend the  use  of  anhydrous  sodium  sulphite  instead  of  the  crystallized 
variety.  Harding  and  Ostenberg23  add  sodium  sulphite  solution  to 
a  measured  amount  of  .5  per  cent  fuchsin  to  determine  the  propor- 

•lEndo,  Cent.  f.  Bakt.,  xxxv,  1904. 

-Kastle  and  Elvove,  Jour.  Inf.  Dis.,  xvi,  1909. 

23  Harding  and  Ostenberg,  Jour,  of  Inf.  Dis.,  xi,  1,  1909. 


160  BIOLOGY  AND  TECHNIQUE 

tions  which  give  the  greatest  delicacy  of  reaction  as  tested  with 
formaldehyd.  The  proportions  so  determined  are  then  added  to 
the  hot  3  per  cent  agar. 

Although  Endo  described  his  medium  as  dependent  upon  the 
formation  of  acid  by  the  bacteria,  this  is  not  so.  Acids  give  no 
coloration  of  the  sulphite-fuchsin  mixture.  Indeed  this  mixture  is 
used  by  chemists  under  the  name  of  Schiff's  reagent  as  a  test  for 
aldehyds.  Acids  decolorize  the  red  caused  by  aldehyds,  and  this 
accounts  for  the  frequent  late  discoloration  of  red  colon  colonies  on 
prolonged  cultivation.  The  medium  is  red  when  hot,  and  colorless 
when  cold,  because  the  compound  between  sulphite  and  fuchsin  dis- 
sociates in  the  hot  solution. 

Robinson  and  Rettger's  Modification  of  Endo.24 — This  seems  to  be  at 
present  the  most  useful  modification  of  Endo  available. 

1.  To  1  liter  of  water  add  25  grams  of  agar,  10  grams  of  pepton,  and 
5  grams  of  meat  extract.  Dissolve  the  agar,  meat  extract  and  pepton.  Bring 
to  P  H  6.8,  and  heat  in  autoclave  for  l/2  hour  at  15  pounds.  Filter  through 
cotton. 

To  this  add  10  c.c.  of  a  10  per  cent  sodium  carbonate  solution.  Heat 
for  a  few  minutes,  and  add  1  per  cent  lactose.  The  fuchsin  sulphite  indicator 
is  then  added  in  the  form  of  5  c.c.  of  saturated  alcoholic  fuchsin,  and  10  c.c. 
of  a  10  per  cent  solution  of  sodium  bisulphite.  This  is  tubed  and  sterilized. 
Krumwiede  recommends  preparing  the  medium  by  adjusting  the  reaction 
to  PH  8.5  in  the  first  place,  relying  upon  sterilization  and  the  addition  of 
the  bisulphite  to  bring  the  reaction  to  the  desired  end  point. 

The  best  results  are  obtained  by  adding  the  lactose,  fuchsin  and  bisulphite 
just  before  use,  and  this  can  be  done  most  conveniently  if  the  agar  basis 
is  bottled  in  100  c.c.  amounts.  The  final  reaction  of  the  medium  should  be 
P#  8.  A  more  acid  reaction  favors  the  diffusion  of  the  indicator.  In  our 
own  laboratory  we  have  found  that  the  addition  of  these  amounts  of  fuchsin 
and  sodium  bisulphite  to  the  medium  inhibit  the  growth  of  typhoid  in  some 
instances.  We  have  obtained  equally  good  differentiation  by  using  0.25  per 
cent  fuchsin  instead  of  0.5  per  cent,  and  0.5  per  cent  sodium  bisulphite 
instead  of  1  per  cent. 

Kendall's  Modification  of  Endo's  Medium.2^ — 1.5  per  cent  meat  extract 
agar  is  prepared,  and  the  reaction  adjusted  faintly  alkaline  to  litmus  by 
the  addition  of  NaOH.  This  agar  is  stored  in  small  flasks  and  it  is  usually 
convenient  to  keep  flasks  containing  100  c.c.  each.  Just  before  use,  1  per 
cent  of  lactose  is  added,  and  then  decolorized  fuchsin  solution,  as  in  Endo's 


2*Kobinson  and  Eettger,  Jour,  of  Med.  Kes.,  24,  1916,  363. 
28  Kendall,  Boston  Med.  &  Surg.  Jour. 


THE   PREPARATION   OF   CULTURE   MEDIA  161 

medium.  Add  about  1  c.c.  of  decolorized  fuchsin  solution,  made  up  as  above 
by  mixing  roughly  prepared  10  per  cent  sodium  sulphite  with  saturated 
alcoholic  fuchsin.  (The  proportions  of  fuchsin  and  sulphite  are  sometimes 
difficult  to  adjust,  possibly  by  reason  of  impurities  in  the  sulphite  due  to 
formation  of  sulphate.  The  instructions  given  by  most  workers  at  present 
are  to  use  10  c.c.  of  a  10  per  cent  aqueous  solution  of  sodium  sulphite, 
and  to  add  to  this  1  c.c.  of  a  10  per  cent  solution  of  fuchsin  in  96  per  cent 
alcohol.)  When  these  flasks  containing  the  various  ingredients  are  hot  they 
are  red  or  pink,  but  when  plates  are  poured  and  allowed  to  harden,  the 
medium  should  be  either  colorless  or  very  faintly  pinkish.  It  is  best  to  pour 
a  number  of  plates  rather  thickly  and  then  allow  them  to  dry  with  the 
covers  off.  Inoculations  from  the  feces  suspension  are  then  made  by  surface 
smear,  with  a  bent  glass  rod.  Colon  colonies  are  pinkish  and  red;  typhoid 
colonies,  smaller  and  grayish. 

In  concluding  the  description  of  some  of  the  most  important  typhoid 
isolation  media,  we  would  like  to  add  that  a  great  deal  seems  to  depend 
upon  the  habit-acquired  skill  which  the  individual  worker  attains.  None  of 
these  stool  isolation  media  are  ordinarily  successful  at  once  in  the  hands 
of  anyone,  and  a  certain  amount  of  practice  must  be  attained  before  one  can 
judge  of  the  usefulness  or  uselessness  of  a  medium. 

Brilliant  Green  Agar  for  Typhoid  Isolation. — Krumwiede  has 'recently 
devised  a  brilliant  green  agar  with  which  he  has  had  excellent  results.26 

The  basis  is  an  extract  agar  like  that  used  for  Endo's  medium : 

Beef  Extract 0.3% 

Salt   0.5% 

Peptone 1.0% 

Agar    1.5% 

(Domestic  peptones  are  satisfactory.) 

Dissolve  in  autoclave;  clear  and  filter.  A  clear  agar  is  essential.  The 
final  reaction  of  the  medium  is  to  be  neutral  to27  Andrade's  indicator,  which 
in  terms  of  phenolphthalein  is  0.6-0.7%  acid  (normal  HC1)  or  P^  7.2.  It  is 
more  convenient  to  have  the  reaction  set  slightly  alkaline  to  litmus  at  the  time 
of  preparation  and  to  acidify  each  bottle  as  used.  The  agar  is  bottled  in 
100  c.c.  amounts  and  autoclaved.  When  needed,  the  bottles  are  melted  and 
the  volume  of  each  corrected  (if  necessary)  to  an  approximate  100  c.c..  Add 
to  each  bottle: 


28  We  are  indebted  to  Dr.  Krutnwicde  for  a  preliminary  account  of  this  method. 

21  Andrade's  Indicator:  0.5  per  cent  aqueous  acid  fuchsin 100  c.c. 

Normal  NaOH 16  c.c. 

The  dye  is  slowly  (2  hours)  alkalinized  to  the  color-base;  the  red  tint  is  restored 
by  acids. 


162  BIOLOGY   AND   TECHNIQUE 

One  per  cent  Andradc  Indicator. 

Acid  to  bring  to  neutral  point  of  the  indicator.28 

One  per  cent  Lactose.29 

0.1  per  cent  Glucose. 

Brilliant  Green  in  0.1  per  cent  aqueous  solution. 

Two  dilutions  of  dye  are  •  used  in  routine  plating,  corresponding  to 
1-500,000  and  1-330,000  in  terms  of  solid  dye  (0.2  c.c.  and  0.3  c.c.  of  0.1 
per  cent  solution  per  100  c.c.  of  agar).  The  sample  of  dye  which  Krumwiede 
has  used  is  from  Bayer,  but  he  has  also  tested  and  found  equally  satisfactory 
samples  from  Griibler  and  Hochst.  0.1  gram  of  dye  is  accurately  weighed 
on  a  foil,  washed  with  boiling  H20  into  a  100  c.c.  volumetric  flask  and  made 
up  to  the  mark  when  cool.  The  flask  should  be  clean  and  neutral  (by  test). 
Fresh  solutions  vary  in  activity  (see  standardization  tests) ;  they  keep  about 
a  month. 

Each  bottle  is  mixed  and  poured  into  six  plates  only  (a  thick  layer  of 
agar  gives  the  most  characteristic  colonies).  Plates  are  left  uncovered  until 
agar  has  "jellied";  porous  tops  are  used;  dry  plates  are  essential  to  avoid 
diffusion. 

Standardization:  The  agar  must  have  proper  "balance."  The  reaction  is 
important;  sediment  reduces  the  activity  of  the  dye  and  light  colored  media 
are  better  than  darker  ones.  Different  lots  of  agar  with  the  same  dye  solution 
act  ununif ormly ;  a  new  batch  or  a  new  solution  must  be  tested. 

Any  variation  in  the  composition  of  the  media  necessitates  a  readjustment 
of  dye  concentration;  this  statement  cannot  be  over-emphasized. 

Brilliant  green,  in  appropriate  dilutions,  not  only  inhibits  all  Gram- 
positive  and  many  Gram-negative  bacteria,  but  exhibits  differential  action  on 
the  colon-typhoid  group.  Paratyphoid  and  the  B.  lactis  aerogenes  are 
untouched,  typhoid  is  restrained  only  at  low  dilutions,  while  dysentery  and 
the  other  colon  group  are  extremely  susceptible.  The  typhoid  colony  on 
this  medium  is  characteristic.  Looking  through  the  plate  against  a  dark 
surface,  in  oblique  light  the  colony  has  a  snowflake  appearance;  the  edge 
delicately  serrate.  With  artificial  light  and  a  hand  lens,  the  texture  is  that 
of  a  coarse  woolen  fabric.  Acid  production  from  the  trace  of  glucose  may 
tinge  the  colony.  The  colony  is  large. 

Brilliant  Green-Eosin  Agar.30 — Meat  infusion  agar  is  prepared  and 
titrated  to  +1  to  phenolphthalein.  The  following  substances  are  added: 


28  An  agar  is  neutral  to  Andradc  when,  hot,  the  color  is  a  deep  red,  but  fades 
completely  on  cooling.     This  is  determined  by  cooling  3  or  4  c.c.  of  acidified  hot 
agar  in  a  serum  tube  under  the  tap  and  adjusting  accordingly, 

29  These    are    conveniently    added    from    one    sterile    solution    containing    20% 
lactose  and  2%  dextrose,  5  c.c.  to  100  of  agar  gives  the  requisite  concentration. 

30  Teague  and  Clurman,  Jour,  of  Infcc.  Dis.,  18,  1916,  647. 


THE   PREPARATION   OF  CULTURE   MEDIA  163 

Eosin 3/50     per  cent 

Brilliant  green    1/300  per  cent 

Saccharose    1  per  cent 

Lactose  1  per  cent 

The  typhoid  colonies  on  this  medium  are  large  and  have  a  grayish  pink 
color.  Most  strains  of  B.  coli  do  not  grow  upon  it;  the  colonies  of  B.  coli 
that  do  develop  have  deep  red  centers.  Meyer  and  Stickel31  claim  that 
better  results  are  obtained  with  the  brilliant  green-eosin  medium  if  peptic 
digest  agar  is  substituted  for  the  meat  infusion  agar.  They  set  their  reaction 
at  Pff  =7  to  6.8. 

Malachite-Green  Bouillon  (Peabody  and  Pratt).32— To  100  c.c.  of  beef 
infusion  broth  add  10  c.c.  of  one  per  cent  solution  of  malachite  green  Hochst 
120,  made  with  sterile  water.  This  is  tubed. 

This  medium  is  used  as  an  enriching  fluid.  One  drop  of  the  suspected 
material  (emulsified  stool)  is  added  to  each  tube  and  after  incubation  for 
eighteen  to  twenty-four  hours  inoculations  may  be  made  upon  plates. 

Peabody  and  Pratt  found  a  reaction  of  .5  per  cent  acidity  to  phenol- 
phthalein  most  favorable. 

Lead  Acetate  Agar  for  the  Differentiation  of  Paratyphoid  "A"  and  "B."  33 
— One  drop  of  a  ten  per  cent  solution  of  neutral  lead  acetate  is  added  to 
every  4  c.c.  of  agar.  This  is  the  original  procedure  of  Burnet  and  Weissen- 
bach.  Krumwiede  recommends  the  cooling  of  the  agar  to  60°,  then  adding 
enough  of  a  0.25  per  cent  basic  lead  acetate  solution  to  bring  the  concentration 
to  0.05  per  cent.  The  agar  is  tubed,  and  Burnet  and  Weissenbach  recommend 
inoculating  with  a  fine  needle  in  several  places  between  the  agar  an_d  the 
walls  of  the  tube.  Typhoid  and  paratyphoid  "B"  bacilli  blacken  the  medium, 
while  paratyphoid  "A"  leaves  it  unchanged.  B.  Enteritidis  and  Typhi 
Murium  behave  like  paratyphoid  "B." 

Bile  Medium?* — (Recommended  for  blood  cultures  by  Buxton  and  Cole- 
man.)  The  medium  is  prepared  as  follows: 

Ox-bile    900  c.c. 

Glycerin   100  c.c. 

Pepton   20  grams 

.t  into  small  flasks  containing  quantities  of  about  100  c.c.  .each  and  sterilized 
;  fractional  sterilization. 
Jackson's  Lactose-Bile  Medium.™ — This  medium  is  used  for  isolating  B. 


31  Meyer,  K.  F.,  and  Sticlce I,  J.  K.,  Jour,  of  Infec.  Dis.,  23,  1918,  48. 
3-  Peabody  and  Pratt,  Boston  Med.  and  Surg.  Jour.,  clviii,  7,  1008. 

33  Burnet  and  Weissenbach,  C.  R.  de  la  Soc  de  Biol.,  vol.  78,  1915,  p.  565. 

34  Conradi,  Dent.  med.  Woch.,  32,  1906. 

35JacJcson,  "Biol.  Studies  of  Pupils  of  W.  T.  Sedgwick,"  1906,  Univ.  Chicago 
?ress. 


164  BIOLOGY  AND  TECHNIQUE 

typhosus  and  B.  coli  from  water,  and  serves  as  a  valuable  enriching  medium 
in  isolating  them  from  other  sources.  Jackon  and  Melia36  found  that  in 
this  medium  B.  typhosus  and  B.  coli  outgrow  all  other  microorganisms  and 
eventually  B.  typhosus  will  even  outgrow  B.  coli. 

It  consists  of  sterilized  undiluted  ox-bile  (or  a  ten  per  cent  solution 
of  dry,  fresh  ox-bile)  to  which  is  added  one  per  cent  pepton  and  one  per 
cent  lactose.  It  is  filled  into  fermentation  tubes  and  sterilized  by  the  frac- 
tional method. 

MacConkey's  Bile-Salt  Agar: 

Sodium  glyeocholate 0.5  per  cent. 

Pepton    2.0    "       " 

Lactose  1.0    ' '      " 

Agar    '. ...    1.5    "      " 

Tap  water q.8. 

The  agar  and  pepton  are  dissolved  and  cleared  and  the  lactose  and  sodium 
glyeocholate  added  before  tubing.  The  B.  typhosus  produces  no  change; 
B.  coli,  producing  acid,  causes  precipitation  of  the  bile  salts. 

Neutral-Red  Medium. — To  100  c,c.  of  a  one  or  two  per  cent  glucose 
agar  add  1  c.c.  of  a  saturated  aqueous  solution  of  a  neutral-red. 

The  medium  is  used  in  tubes,  stab  or  shake  cultures.  The  typhoid  bacillus 
produces  no  change,  while  members  of  the  colon  group  render  the  medium 
colorless  by  reduction  of  the  neutral-red  and  produce  gas. 

Bariekow's  Medium*1 — To  200  c.c.  of  cold  water,  add  10  grams  of 
nutrose  and  allow  to  soak  for  one-half  to  one  hour.  Pour  this  into  800 
c.c.  of  boiling  water,  and  heat  for  twenty  minutes  in  an  Arnold  sterilizer 
at  100°  C.  Filter  through  cotton  and  to  the  opalescent  solution  of  nutrose 
add  5  grams  of  NaCI,  10  grams  of  lactose,  and  sufficient  aqueous  litmus 
solution  to  give  a  pale  blue  color.38 

Russell's  Double  Sugar  Agar.^ — Russell  has  devised  a  simple  medium  for 
quick  identification  of  typhoid  bacilli. 

A  2%  or  3%  extract  agar  is  used,  about  0.8%  acid  to  phenolphthalein. 
Enough  litmus  solution  is  added  to  give  it  the  ordinary  deep  blue.  The 
reaction  is  then  adjusted  with  sodium  hydrate  until  neutral  to  litmus.  Finally 
1%  lactose  and  0.1%  glucose  (dissolved  in  a  small  amount  of  hot  water)  are 
added,  the  medium  is  carefully  sterilized  in  the  Arnold  sterilizer  and  slanted. 
Inoculations  are  made  by  surface  streak  and  stab. 


36  Jackson  and  Melia,  Jour.  Inf.  Dis.,  vi,  1909. 

87  BarsieTcow,  Wien.  klin.  Bund.,  xliv,  1901. 

38  Filtration  may  be  done  through  paper,  but  takes  a  long  time. 

*gKussell,  Jour.  Med.  Kesearch,  xxv,  1911,  217. 


THE   PREPARATION  OF  CULTURE   MEDIA  165 

Sharper  results'  are  obtained  with  this  medium  if  1  per  cent  of  the 
Andrade  indicator  (described  above),  is  substituted  for  the  litmus.  When 
Andrade  is  used,  the  final  reaction  of  agar  should  be  about  P^  7.2.  It  ib 
best  to  standardize  the  reaction  against  the  particular  solution  of  Andrade 
used.  The  reaction  of  the  medium  is  satisfactory  when  the  mixture  containing 
1  per  cent  of  the  indicator  is  red  when  hot,  and  colorless  when  cold.  Typhoid 
,and  paratyphoid  A  and  B,  and  dysentery,  on  this  medium  show  colorless 
growths  on  the  slant,  the  butts,  however,  where  partial  anaerobiosis  exists, 
show  a  deep  red  color,  due  to  acid  fermentation.  Typhoid  may  be  dis- 
tinguished from  paratyphoid  A  and  B  by  the  fact  that  typhoid,  since  it 
ferments  glucose  without  the  production  of  gas,  forms  no  gas  bubbles  in 
the  butt,  whereas,  there  is  gas  formation  with  paratyphoid  A  and  B.  Typhoid 
and  dysentery  give  the  identical  reaction  on  Russell's  medium  colorless  growth 
on  the  slant,  and  acid  formation  without  gas  in  the  butt,  and  must  be 
distinguished  by  the  motility  test.  There  is  no  reliable  way  of  distinguishing 
between  paratyphoid  A  and  B,  unless  lead  acetate  is  added  to  the  medium  (see 
below).  Krumwiede  recommends  the  addition  of  1  per  cent  saccharose  to 
the  1  per  cent  lactose,  and  0.1  per  cent  glucose  as  a  means  of  ruling  out 
some  .of  the  "paratyphoid-like"  intermediates  that  ferment  saccharose  more 
rapidly  than  lactose.  The  non-pathogenic  types  fermenting  lactose,  or  lactose 
and  saccharose,  which  are  present  in  1  per  cent  concentrations,  produce  acid 
on  the  slant,  as  well  as  in  the  butt,  with  the  formation  of  gas^  and  are  thus 
easily  eliminated. 

Russell  Double  Sugar  Agar  with  Lead  Acetate.40 — The 'best  basis  for  this 
medium  is  an  infusion  agar  which  has  been  rendered  sugar  free  and  adjusted 
to  P^  7.4,  or  neutrality  to  Andrade  indicator.  To  this  medium  add  1  per 
cent  Andrade  indicator,  tube  and  sterilize  in  quantities  of  5  c.c.  to  each  tube. 

Make  up  a  solution  containing  20  per  cent  lactose  and  2  per  cent  glucose. 
Sterilize. 

Make  up  a  solution  of  0.25  basic  lead  acetate.     Sterilize. 

To  each  tube  of  the  agar  add  0.25  c.c.  of  the  double  sugar  solution  and 
1  c.c.  of  the  lead  acetate  solution. 

Add  both  the  solutions  to  the  agar  at  60°  C.  under  sterile  precautions 
and  slant. 

Typhoid  bacilli  cause  a  brown  color  near  the  surface  of  the  stab.  Para- 
typhoid "A"  and  dysentery  do  not  cause  any  browning.  Paratyphoid  "B" 
and  other  members  of  the  group  cause  browning.  (The  volume  of  agar  per 
tube  is  not  given  in  the  article,  but  the  sugar  percentage  works  out  for  5  c.c.) 

40  Kligler,  Jour,  of  Experimental  Medicine,  September,  1918. 


166  BIOLOGY  AND  TECHNIQUE 

• 

SPECIAL  MEDIA  FOR  THE  ISOLATION  OF  CHOLERA  SPIRILLA 

Dieudonnc  Medium.** — To  70  parts  of  ordinary  3  per  cent  agar  made 
neutral  to  litmus,  add  30  parts  of  a  sterile  mixture  of  defibrinated  beef 
blood  and  normal  sodium  hydrate. 

This  is  sterilized  by  steam  before  being  added  to  the  agar.  This  alkali 
agar  is  poured  into  plates  and  allowed  to  dry  several  days  at  37°,  or  5  minute* 
at  60°.  The  material  to  be  examined  is  smeared  on  the  surface  of  these 
plates  with  a  glass  rod. 

Aronson's  Medium  for  Cholera  Stool  Isolation.42 — This  medium  is  pre- 
pared as  follows:  35  grams  of  agar  are  added  to  1  liter  of  tap  water  and 
soaked  over  night. 

Add  10  grams  of  meat  extract,  10  grams  of  pepton,  5  grams  of  sodium 
chloride  and  heat  in  steam  sterilizer  from  4  to  5  hours.  The  particles  are 
allowed  to  settle  by  letting  the  hot  agar  stand,  and  the  clear  supernatant 
agar  poured  into  flasks  to  hold  100  c.c.  each. 

The  following  solutions  are  previously  made  and  sterilized  for  l/2  hour  in 
the  Arnold: 

1.  10  per  cent  solution  of  sodium  carbonate 

2.  20  per  cent  solution  of  cane-sugar 

3.  20  per  cent  solution  of  dextrin 

4.  saturated  solution  of  basic  fuchsin 

5.  10  per  cent  solution  of  sodium  sulfite   (sterilized  by  being  brought  to 

a  boil) 

To  100  c.c.  of  agar  add  6  c.c.  of  the  10  per  cent  solution  of  sodium 
carbonate  and  heat  for  15  minutes  at  100°  C.  The  agar,  because  of  the 
alkalinity,  becomes  brown  and  cloudy.  While  hot,  add  5  c.c.  of  the  20  per 
cent  solution  of  cane-sugar,  5  c.c.  of  the  20  per  cent  solution  of  dextrin, 
0.4  c.c.  of  the  saturated  solution  of  basic  fuchsin,  and  2  c.c.  of  the  10  per 
cent  sodium  sulfite  solution.  The  flask  is  allowed  to  stand  to  let  the  coarser 
particles  settles,  and  plates  are  poured  with  the  clear  supernatant  fluid.  The 
principle  of  this  medium,  like  Dieudonne's,  depends  upon  the  ability  of 
cholera  spirilla  to  grow  on  very  alkalin  media  and  upon  their  ability  to 
split  polysaccharites  with  acid  and  aldehyde  formation. 

Cholera  strains,  recently  from  the  human  body,  give  large  red  colonies 
in  from  15  to  20  hours,  whereas,  the  colon  colonies  are  smaller  and  colorless. 
Teague  and  Travis43  found  that  strains  of  cholera  spirillum  that  had  been 
out  of  the  human  body  for  some  time  did  not  yield  red  colonies  promptly, 
but  they  obtained  excellent  results  even  with  these  cultures,  if  they  added 
0.25  per  cent  nutrose  to  Aronson's  medium. 

"Dieudonne,  Cent.  f.  Bakt.,  1.,  orig.,  1909. 

"Aronson,  Deut.  med.  Woch.,  41,  1915,  1027. 

43  Teague  and  Travis,  Jour,  of  Infec.  Dis.,  18,  1916,  601. 


THE   PREPARATION   OF  CULTURE   MEDIA  167 

Teayue  and  Travis  Medium  for  the  Cholera  Spirillum.** — They  prepare 
their  medium  as  follows: 

Two  pounds  of  chopped  beef  are  soaked  in  2  liters  of  distilled  water  in 
the  ice  box  over  night.  The  fluid  is  squeezed  out,  heated  in  the  Arnold, 
filtered  through  filter  paper  and  made  neutral  to  litmus  with  sodium  hydrate, 
and  is  heated  again.  After  being  allowed  to  cool,  it  is  inoculated  with  colon 
bacillus,  and  incubated  for  2  or  3  days  to  make  it  sugar  free.  Agar  is  then 
prepared  from  it  by  adding  1  per  cent  pepton,  0.5  per  cent  sodium  chlorid 
and  clearing  with  egg.  The  reaction  is  adjusted  to  —0.5  phenolphthalein, 
and  after  the  agar  has  been  cleared  and  filtered,  0.25  per  cent  nutrose  is 
added.  A  stock  aqueous  solution  of  3  per  cent  bluish  eosin  is  kept  on  hand 
in  the  dark,  also  a  1  per  cent  stock  solution  of  Bismarck-brown.  The  Bis- 
marck-brown solution  must  be  made  up  in  water  containing  10  per  cent  of 
lactose,  because  it  is  entirely  soluble  to  1  per  cent  in  distilled  water  alone. 

To  50  c.c.  of  this  nutrose  agar  add  1  per  cent  saccharose,  1  c.c.  of  the 
3  per  cent  eosin  solution,  and  2  c.c.  of  the  1  per  cent  Bismarck-brown  solution. 
After  this  mixture  has  been  shaken  until  the  stains  are  uniformly  distributed, 
pour  plates.  These  plates  are  uncovered  and  placed  on  a  shelf,  face  down, 
in  the  incubator  for  20  minutes  to  remove  excess  of  moisture  before  smearing. 
On  this  medium  the  differentiation  of  the  cholera  colonies  is  striking,  with 
large  and  with  red  centers,  while  the  colon  colonies  are  uniformly  pink. 

Rabbit's  Blood  for  Ducrey  Bacillus  Cultivation. — Rabbits  are  bled  from 
the  heart  with  a  sterile  syringe  and  about  IVk  to  2  c.c.  placed  into  small 
test  tubes.  The  blood  is  allowed  to  clot  and  inactivated  at  56°  for  y%  hour. 
This  makes  an  excellent  medium  for  the  cultivation  of  the  Ducrey  bacillus, 
for  streptococci  and  some  other  organisms.  It  is  also  excellent  for  the 
preservation  of  streptococci  and  pneumococci  in  a  virulent  condition. 

The  preparation  of  Anaerobic  Tissue  Tubes  for  the  Cultivation  of  Spiro- 
chaetes  and  other  Anaerobes. — This  is  the  Tarrozi  and  Smith  method  of 
using  tissue  for  anaerobic  purpose,  adapted  by  Noguchi  for  the  cultivation 
of  various  spirochaetes.  The  proportions  of  broth,  serum  and  agar  are 
adapted  to  the  particular  purpose  for  which  it  is  to  be  used.  It  is  necessary, 
therefore,  in  this  place  only  to  describe  the  best  method  of  putting  up  the 
tissue  tubes.  High,  narrow  test  tubes  are  used,  about  7  to  8  inches  in  length, 
with  a  diameter  not  larger  than  that  of  a  Wassermann  tube.  One-half  inch 
tubes  are  convenient.  A  rabbit  is  rapidly  killed  by  ether  anesthesia.  It  is 
best  to  bleed  him  from  the  carotid  at  the  same  time,  in  order  not  to  waste 
the  blood.  The  animal  is  immediately  opened  with  sterile  precautions.  It 
is  best  to  dissect  off  the  fur  and  wash  the  abdominal  wall  with  alcohol  before 
opening  the  abdomen.  The  abdomen  is  then  opened  carefully  and  widely, 
so  that  tlio  organs  can  T>c  easily  reached  without  unnecessary  poking  about. 


44  Teague  ami  Travis,  Jour,  of  Infec.  Dis.,  18,  1916,  601. 


168  BIOLOGY  AND  TECHNIQUE 

The  intestines  are  pulled  aside  so  as  to  uncover  the  kidneys.  With  a  fresh 
set  of  instruments,  the  hilum  of  the  kidney  is  now  grasped  and  the  kidney 
rapidly  separated  from  its  capsule  and  passed  through  the  flame  before 
being  placed  into  a  sterile  Petri  plate.  The  spleen  may  be  removed  in  the 
same  way. 

The  kidney  and  spleen  should  then  be  cut  up  with  the  utmost  precautions 
of  sterility.  Work  in  a  dustless  place  with  the  windows  closed  and  several 
Bunsen  flames  going  close  to  the  field  of  operation.  An  assistant  slightly 
raises  the  cover  of  the  Petri  plate  and  the  bacteriologist,  working  with  sterile 
forceps  and  a  sterile  old  knife  which  can  be  constantly  flamed,  cuts  the 
kidney  in  pieces  of  appropriate  size  against  the  bottom  of  the  plate.  In 
placing  these  bits  of  tissue  into  tubes,  the  stopper  of  the  sterilized  tube 
is  pulled,  and  the  tube  heated  around  its'  lips  and  upper  one-half  inch. 
The  tissue  is  then  rapidly  passed  into  the  flame,  thrust  into  the  mouth  of 
the  tube,  the  cotton  stopper  flamed  and  inserted.  The  tube  is  then  given 
a  rapid  flip  with  the  hand,  which  sends  the  tissue  to  the  bottom.  When 
these  tubes  are  filled  with  broth  and  ascitic  fluid  or  agar,  they  should  be 
incubated  before  use  and  the  unsterile  ones  discarded. 

In  most  of  Noguchi's  work,  and  some  of  our  own,  paraffin  oil  was  used 
over  the  tops  of  these  tubes.  The  sealing  properties  of  this,  however,  are 
not  what  they  were  formerly  supposed  to  be.  Air  passes  through  this 
paraffin  oil,  and  if  sealing  is  desired  it  is  much  better  to  heat  the  upper 
empty  part  of  the  tubes  thoroughly,  and  thrust  in  a  paraffin  stopper.  The 
top  of  the  fluid  also  can  be  covered  with  melted  paraffin  which  will  solidify 
in  the  incubator. 

Cooked  Meat  Medium,  Robertson,45  for  the  Cultivation  of  Anaerobes. — 
250  grams  of  beef  heart  are  minced  and  ground  in  a  mortar.  Add  250  c.c. 
of  tap  water,  heat  slowly,  cook  thoroughly,  neutralize  to  litmus  with  NaOH, 
tube  and  sterilize  in  autoclave. 

The  simplest  method  of  making  cooked  meat  media  which  gives  satis- 
factory results  with  the  majority  of  the  anaerobic  bacilli,  is  the  following. 
A  few  pieces  of  chopped  meat  (not  necessarily  heart)  are  placed  in  the 
bottom  of  the  tube,  enough  infusion  broth,  PH  7.8,  is  added  so  that  there 
are  about  3  c.c.  clear  broth  over  the  meat.  The  medium  is  ready  for  use 
after  autoclaving.  The  reaction  usually  becomes  more  acid  on  autoclaving 
in  the  presence  of  the  meat  fragments.  The  optimum  for  the  most  anaerobic 
bacilli  is  PH  =7.4. 

Enriching  Substances  Used  in  Media. — For  the  cultivation  of 
microorganisms  which  are  sensitive  to  their  food  environment,  it  is 
often  necessary  or  advisable  to  add  to  the  ordinary  media  enriching 
substances,  which  empirical  study  has  shown  to  favor  the  growth 


ts  Robertson,  Jour,  of  Path,  and  Bacter.,  January,  1916,  20,  No.  3. 


THE  PREPARATION  OF  CULTURE   MEDIA  16§ 

of  the  organism  in  question.  The  substances  most  commonly  used 
for  such  enrichment  are  glucose,  nutrose  (sodium  casemate),  gly- 
cerin, sodium  formate,  and  uiisolidified  animal  proteins.  As  animal 
and  blood  serum  and  whole  blood  must  frequently  be  used  in  this 
way,  an  understanding  of  the  methods  employed  in  obtaining  these 
substances  is  necessary. 

Method  of  Obtaining  Blood  and  Blood  Media. — Blood  serum  from 
beef  and  sheep  may  be  collected  in  the  manner  recommended  for 
the  collection  of  such  serum  in  the  preparation  of  Loeffler's  medium, 
pipetted  into  test  tubes,  and  sterilized  in  the  fluid  state  by  exposure 
to  temperatures  ranging  from  60°  to  65°  C.,  for  one  hour  upon  six 
consecutive  days.  It  is  not  a  simple  matter  to  sterilize  serum  in 
this  way  and  requires  much  time  and  care. 

The  method  most  commonly  employed,  in  laboratories  which  have 
access  to  hospitals,  for  obtaining  clear  serum  depends  upon  the  col- 
lection of  exudate  or  transudate  fluids  by  sterile  methods  directly 
from  the  pleural  cavity,  the  abdominal  cavity,  or  the  hydrocele 
cavity.  Sterile  flasks  or  test  tubes  are  prepared  and  the  fluid  is 
allowed  to  flow  directly  out  of  the  cannula  into  these.  It  is  necessary 
to  avoid  carbolic  acid  or  other  disinfectants  in  sterilizing  instru- 
ments and  rubber  tubing  used  during  the  operation.  These  should 
be  brought  into  the  ward  in  the  water  in  which  they  have  been 
boiled  and  not  in  strong  antiseptic  solutions,  as  is  frequently  done. 
The  fluid  so  obtained  may  be  incubated  and  the  contaminated  tubes 
discarded.  The  serum  may  then  be  added,  in  proportions  of  one  to 
three,  to  sterile  broth  or  melted  agar. 

Agar  thus  used  is  melted  and  cooled  to  60°  C.,  or  below.  One- 
third  of  its  volume  of  warmed  exudate  fluid  is  added,  and  the  plates 
are  poured. 

Serum  may  be  rendered  free  of  bacteria  by  filtration  through  a 
Berkefeld  or  Pasteur-Chamberland  filter.  This  is  an  effectual 
method,  but  requires  much  time  and  care. 

Whole  blood  may  be  obtained  for  cultural  purposes  by  bleeding 
rabbits  or  dogs  or  other  animals  directly  from  a  blood-vessel  into 
tubes  of  melted  agar.  In  the  case  of  a  rabbit,  after  the  administra- 
tion of  an  anesthetic  (ether),  an  incision  is  made  directly,  over  the 
trachea,  and,  by  careful  section,  the  carotid  artery  is  isolated,  lying 
close  to  the  side  of  the  trachea.  Blood  may  be  collected  and 
hemolysed  by  the  gradual  addition  of  the  smallest  amount  of  ether 
which  will  completely  hemolyse  the  amount  treated.  This  may  be 


170  BIOLOGY  AND  TECHNIQUE 

kept  in  stoppered  sterile  bottles  and  added  to  agar  as  desired.  This 
is  particularly  useful  in  preserving  blood  for  routine  work  on  menin- 
gococcus  carriers. 

Methods  of  bleeding  animals  are  briefly  described  above. 

SELECTIVE  ACTION  OF  DYE  STUFFS 

In  describing  the  selective  media  for  typhoid  bacilli  we  have 
seen  that  malachite  green  and  crystal  violet  have  been  found  to 
exert  a  certain  amount  of  selective  action  upon  the  typhoid  and 
colon  groups.  The  selective  influence  of  various  dyes  has  been 
recently  again  studied  by  Churchman.  Churchman46  found  that  the 
addition  of  gentian  violet  in  dilutions  of  1 :100,000,  to  media,  in- 
hibited some  bacteria,  while  others  grew  luxuriantly  in  its  presence. 
Extremely  interesting,  both  practically  and  theoretically,  is  his  ob- 
servation that  upon  such  gentian  violet  media  bacteria  fall  into  two 
groups.  Those  which  grow  on  gentian  violet  correspond  in  a  general 
way  to  the  Gram-negative  bacteria;  those  which  fail  to  develop  on 
these  media  correspond  roughly  with  the  Gram-positive  species. 
One  strain  of  the  enteritidis  group  could  not  be  cultivated  on  gen- 
tian violet,  and  this  was  found  to  differ  from  the  others  also  in  its 
agglutination  tests. 

Signorelli47  claims  that  dahlia  is  useful  in  differentiating  true 
cholera  strains  from  similar  spirilla.  The  true  cholera  strains  grew 
with  colored  colonies,  while  others  remain  colorless,  in  his  experi- 
ments. 

Krumwiede  and  Pratt48  were  unable  recently  to  confirm  the 
claims  of  Signorelli.  However  they  fully  confirm  the  findings  of 
Churchman  both  as  to  the  selective  action  of  gentian  violet  and  in 
regard  to  the  classification  of  bacteria  into  two  groups  corresponding 
to  their  reaction  to  the  Gram  stain.  They  state  that  among  Gram- 
negative  bacteria  a  strain  is  occasionally  found  which  will  not  grow 
on  the  gentian  violet  media,  differing  in  this  respect  from  other 
members  of  the  same  species.  They  find  also  that  the  reaction  is 
quantitative. 

The   streptococcus-pneumococcus   group,  according  to  their  in- 

*•  Churchman,  Jour.  Exp.  Med.,  16,  1912 ;  also  Churchman  and  Michael,  ibid. 
"Signorelli,  Centralbl.  f.  Bakt.,  Orig.  56,  1912. 

"Krumwiede  and  Pratt,  Centralbl.  f.  Bakt.,  Orig.  68,  1913;  and  Proc.  N.  Y. 
Path.  Soc.,  xiii,  1913. 


THE  PREPARATION   OF  CULTURE   MEDIA  171 

vestigations,  differs  from  other  bacteria  in  being  able  to  grow  in 
the  presence  of  quantities  of  violet  which  inhibit  other  Gram-positive 
species.  Dysentery  bacilli  show  variations.  They  found,  however 
that,  in  addition  to  gentian  violet,  Hoffman  violet,  crystal  violet, 
dahlia  violet,  fuchsin,  rosanilin  and  methyl  violet  will  inhibit  Gram- 
positive  but  not  Gram-negative  bacteria  in  dilutions  of  from  1  to 
5000  to  1  to  50,000. 


CHAPTER   VIII 

METHODS  USED  IN  THE  CULTIVATION  OF  BACTEEIA 

INOCULATION    OF    MEDIA 

THE  transference  of  bacteria  from  pathological  material  to  media, 
or  from  medium  to  medium,  for  purposes  of  cultivation,  is  usually 
accomplished  by  means  of  a  platinum  wire  or  loop.  The  platinum 
wire  used  should  be  thin  and  yet  possess  a  certain  amount  of  stiff- 
ness and  be  about  two  to  three  inches  in  length.  This  is  fused  into 
the  end  of  a  glass  rod  six  to  eight  inches  long.  It  is  an  advantage, 
though  not  necessary,  to  use  rods  of  so-called  "sealing-in"  glass 
which,  having  the  same  coefficient  of  expansion  as  platinum,  does 
not  crack  during  sterilization.  For  work  with  fluid  media,  the  wire 
should  be  bent  at  its  free  end  so  as  to  form  a  small  loop  which 
will  pick  up  a  drop  of  the  liquid.  For  the  inoculation  of  solid  media 
and  the  making  of  stab  cultures,  a  straight  "needle"  or  wire  should 
be  used.  Other  shapes  of  these  wires  and  spatulae  from  heavy  wire 
have  been  devised  for  various  purposes  and  are  easily  improvised 
as  occasion  demands. 

When  making  a  transfer  from  one  test  tube  to  another,  the  tubes 
should  be  held  between  the  thumb  and  first  and  second  fingers  of 
the  left  hand.  (See  Fig.  12.)  The  plugs  are  then  removed  by 
grasping  them  between  the  small  and  ring  fingers  and  ring  and 
middle  fingers  of  the  right  hand,  first  loosening  any  possible  adhe- 
sions between  glass  and  plugs  by  a  slight  twisting  motion.  The 
platinum  wire  is  held  meanwhile  by  the  thumb  and  index  fingers 
of  the  right  hand  in  the  manner  of  a  pen.  The  wire  is  heated  red 
hot  in  a  Bunsen  flame,  and  is  then  passed  into  the  culture  tube 
without  being  allowed  to  touch  the  glass.  It  is  held  suspended 
within  the  tube  for  a  few  seconds  to  permit  of  cooling  before  touch- 
ing the  bacterial  growth.  The  wire  is  then  allowed  to  touch  lightly 
the  surface  of  the  growth  and  a  small  amount  is  picked  up. 
It  is  then  removed  from  the  tube  without  allowing  it  lo 

172 


METHODS  USED  IN  CULTIVATION  OF  BACTERIA  173 

touch  the  sides  of  the  glass,  and  is  passed  into  the  tube  which  is 
to  be  inoculated.  If  the  tube  contains  a  slanted  medium,  such  as 
agar,  a  light  stroking  motion  from  the  bottom  of  the  slant  to  its 
apex  will  deposit  the  bacteria  upon  the  medium  evenly  along  a 
central  line.  The  needle  may  also  be  plunged  downward  into  the 
substance  of  the  nutritive  material  so  that  in  the  same  tube  both 
surface  growth  and  deep  growth  may  be  observed.  If  a  stab  culture 
is  to  be  made  in  unslanted  agar  or  in  gelatin,  the  needle  is  simply 
plunged  straight  downward  as  nearly  as  possible  along  the  axis  of 
the  medium.  If  a  fluid  medium  is  being  inoculated,  the  wire  should 
be  introduced  only  into  the  upper  part  of  the  liquid  and  the  bacteria 
gently  rubbed  into  emulsion  against  the  side  of  the  glass.  The 


FIG.  12. — TAKING  PLUGS  FROM  TUBES  BEFORE  INOCULATION. 

needle  is  then  removed  from  the  tube,  the  stopper  carefully  replaced, 
and  the  platinum  wire  immediately  sterilized  in  the  flame.  This 
sterilization  of  platinum  needles  after  they  have  been  in  contact 
with  bacteria  should  become  second  nature  to  those  working  with 
bacteria,  since  an  infraction  against  this  rule  may  give  rise  to  serious 
and  widespread  consequences.  In  burning  off  platinum  needles  it 
is  well  to  remember  that  a  part  of  the  glass  rod,  as  well  as  the  wire 
itself,  is  introduced  into  the  tubes  and  may  become  contaminated, 
and  for  this  reason  the  rod  itself,  at  least  in  its  lower  two  or  three 
inches,  should  be  passed  through  the  flame  as  well  as  the  wire.  As 
an  extra  precaution  against  contamination,  the  lips  of  test  tubes  and 
flasks  and  the  protruding  edges  of  cotton  plugs  may  be  passed 
through  the  flame  and  singed. 


174  BIOLOGY  AND  TECHNIQUE 

THE  ISOLATION  OF  BACTERIA  IN  PURE  CULTURE 

It  is  obvious  that  in  many  cases  where  bacteria  are  cultivated 
Irom  water,  milk,  pathological  material,  or  other  sources,  many 
species  may  be  present  in  the  same  specimen.  It  is  likewise  obvious 
that  scientific  bacteriological  study  of  any  bacterium  can  be  made 
only  if  we  obtain  this  particular  species  entirely  apart  from  others, 
in  what  is  known  as  "pure  culture."  The  earliest  methods  for 
accomplishing  this  were  the  methods  of  Pasteur  and  of  Cohn  who 
depended  upon  the  power  of  one  species  to  outgrow  all  others,  if 
cultivated  for  a  sufficient  length  of  time  in  fluid  media.  This  method, 
of  course,  was  inadequate  in  that  it  was  often  purely  a  matter  of 
chance  which  one  of  the  mixture  of  species  was  finally  obtained  by 
itself.  A  later  method,  by  Klebs,  depends  upon  serial  dilution,  in 
test  tubes  of  fluid  media,  by  which  the  eventual  transference  of 
one  germ  only,  to  the  last  tube  was  attempted.  Such  methods,  none 
of  them  of  great  practical  value,  have  been  entirely  displaced  by 
those  made  possible  by  Koch's  introduction  of  solid  media  which 
may  be  rendered  fluid  by  heat. 

The  methods  now  employed  for  the  isolation  of  bacteria  depend 
upon  the  inoculation  of  gelatin  or  agar,  when  in  the  melted  state, 
the  thorough  distribution  of  the  bacteria  in  these  liquids  by  mixing, 
and  the  subsequent  congealing  of  these  media  in  thin  layers.  By 
this  means  the  individual  bacteria,  distributed  in  the  medium  when 
liquid,  are  held  apart  and  separate  when  the  medium  becomes  stiff. 
The  masses  of  growth  or  "colonies"  which  develop  from  these  single 
isolated  microorganisms  are  discrete  and  are  descendants  of  a  single 
organism,  and  can  be  transferred,  by  means  of  a  process  known 
as  "colony-fishing,"  to  fresh  sterile  culture  media. 

Plating. — The  first  method  employed  by  Koch  for  bacterial  isola- 
tions was  one  that  consisted  in  the  use  of  simple  plates  of  glass, 
about  3X4  inches  in  size,  mounted  upon  a  leveling  stand.  A 
wooden  triangle,  supported  upon  three  adjustable  screw-feet,  formed 
the  base  of  this  apparatus.  Upon  this  was  set  a  covered  crystallizing 
dish  which  could  be  filled  with  ice  water.  Upon  the  top  of  this 
rested  the  sterilized  plates  under  a  bell  jar.  By  screwing  up  or 
down  upon  the  supports  of  the  triangle,  leveling  of  the  plate  could 
be  achieved  and  controlled  by  a  spirit-level  placed  at  its  side.  The 
inoculated  gelatin  was  poured  upon  the  plate  and  spread  and  rapidly 


METHODS   USED  IN   CULTIVATION   OF  BACTERIA  175 

cooled  and  hardened  by  the  cold  water  contained  in  the  crystallizing 
dish. 

The  original  method  of  Koch  has  been  modified  considerably  and 
the  method  universally  employed  at  present  depends  upon  the  use 
of  circular  covered  dishes,  the  so-called  Petri  dishes.  These  obviate 
the  necessity  of  a  leveling  stand  and  prevent  contamination  of  the 
plate  when  once  poured.  Each  Petri  dish  plate  consists  of  two 
circular  glass  dishes;  the  smaller  and  bottom  dish  has  an  area  of 
63.6  square  centimeters ;  the  larger  is  used  as  a  cover  for  the  smaller, 
and  forms  a  loosely  fitting  lid.  The  plates  when  fitted  together  are 
sterilized  and  thus  form  a  closed  cell  which,  if  properly  handled, 
may  remain  sterile  indefinitely. 


FIG.  13. — INOCULATING. 

The  technique  for  making  a  pour  plate  for  the  purpose  of  isolating 
bacteria  from  mixed  culture  is  as  follows : 

The  actual  " pouring"  of  plates  is  preceded  by  the  preparation 
of  usually  three  graded  dilutions  of  the  material  to  be  examined. 
For  this  purpose  three  agar  or  gelatin  tubes  are  melted  and,  in  the 
case  of  the  agar,  are  cooled  to  a  temperature  of  about  42°  C.  in  a 
water  bath.  A  platinum  loopful  of  the  material  to  be  examined  is 
transferred  to  one  of  these  tubes.  The  bacteria  are  then  thoroughly 
distributed  throughout  the  melted  gelatin  or  agar  by  alternately 
depressing  and  raising  the  plugged  end  of  the  tube,  giving  it  a 
rotary  motion  at  the  same  time.  This  thoroughly  distributes  the 
bacteria  throughout  the  medium  without  allowing  the  formation  of 
air-bubbles,  Two  loopfuls  of  this  mixture  are  then  transferred  to 
the  second  tube  and.  a  similar  mixing  process  is  repeated.  This 


176  BIOLOGY  AND  TECHNIQUE 

second  tube  contains  the  bacteria  in  much  greater  dilution  than  the 
first  and  the  colonies  which  will  form  in  the  plate  poured  from  this 
tube,  will  be  farther  apart.  A  third  dilution  is  then  made  by  trans- 
ferring five  loopfuls  of  the  mixture  in  the  second  tube  to  the  third. 
This  again  is  mixed  as  before.  The  contents  of  the  tubes  are  then 
poured  into  three  sterile  Petri  dishes.  The  pouring  should  be  done 
with  great  care.  The  cover  of  the  dish  is  raised  along  one  margin 
simply  far  enough  to  permit  the  insertion  of  the  end  of  the  test 
tube,  the  plug  of  which  has  been  removed  and  the  lips  passed,  with 
a  rotary  movement,  through  the  flame.  The  medium  is  poured  into 
the  dish  without  the  lips  of  the  tube  being  allowed  to  touch  either 
the  bottom  or  the  cover  of  the  dish.  The  cover  is  then  replaced 
and  the  medium  allowed  to  harden. 


FIG.  14. — POURING  INOCULATING  MEDIUM  FROM  PETRI  DISH. 

When  agar  has  been  used,  the  dishes  may  be  placed  in  an  in- 
cubator at  37°  C.  It  is  well  to  place  the  plates  upside  down  in 
the  incubator.  This  prevents  the  condensation  water,  squeezed  out 
of  the  agar  during  hardening,  from  collecting  on  its  surface,  and 
forming  channels  for  the  diffuse  spreading  of  bacteria.  The  same 
end  may  be  attained  by  the  use  of  Petri  plates  provided  with  porous 
earthenware  lids,  as  suggested  by  Hill.  Simple  inversion  of  the 
plates,  however,  usually  suffices.  When  gelatin  has  been  used,  the 
plates  are  allowed  to  remain  in  a  dark  place  at  room  temperature 
or  in  a  special  thermostat  kept  at  22° -25°  C. 

Colonies  in  agar,  kept  at  37.5°  C.,  usually  develop  in  eighteen 
to  twenty-four  hours ;  those  in  gelatin  or  agar  at  room  temperature 
in  from  twenty-four  to  forty-eight  hours,  depending  upon  the  species 


METHODS   USED  IN  CULTIVATION  OF  BACTERIA  177 

of  bacteria  which  are  being  studied.  Often  in  the  second  dilution, 
more  frequently  in  the  third,  the  colonies  will  be  found  well  apart 
and  can  then  be  "fished."  The  process  of  "colony-fishing"  is  one 
which  requires  practice  and  should  always  be  done  with  care,  for 
upon  its  success  depends  the  purity  of  the  sub-culture  obtained. 
Colonies  should  never  be  fished  under  the  naked  eye,  no  matter  how 
far  apart  and  discrete  they  may  appear,  since  not  infrequently  close 
to  the  edge  of  or  just  beneath  a  larger  colony  there  may  be  a 
minute  colony  of  another  species  which  may  be  too  small  to  be 
visible  to  the  naked  eye,  but  which,  nevertheless,  if  touched  by 
accident  will  contaminate  the  sub-culture. 

For  proper  "fishing,"  the  Petri  plate  with  cover  removed,  should 
be  placed  upon  the  stage  of  the  microscope  and  examined  with  a 
low  power  objective,  such  as  Leitz  No.  2  or  Zeiss  AA.  The  sterilized 
platinum  needle,  held  in  the  right  hand,  is  then  carefully  directed 
into  the  line  of  focus  of  the  lens,  while  the  small  finger  of  the 
hand  is  steadied  upon  the  edge  of  the  microscope  stage.  When  the 
point  of  the  needle  is  clearly  visible  through  the  microscope,  it  is 
gently  depressed  until  it  is  seen  to  touch  the  colony  and  to  carry 
away  a  portion  of  it,  The  needle  is  then  withdrawn  without  again 
touching  the  nutrient  medium  or  the  edges  of  the  glass  or  the  lens, 
and  transferred  to  a  tube  of  whatever  medium  is  desired.  In  this 
way,  individuals  of  one  colony,  descendants  of  a  single  bacterium 
of  the  original  mixture — are  carried  over  to  the  fresh  medium. 

Esmarch  Roll  Tubes.1 — A  simple  method  of  obtaining  separate  colonies 
is  that  devised  by  von  Esmarch  and  known  as  "roll-tube ' '  cultivation.  Tubes 
of  melted  gelatin  are  inoculated  with  various  dilutions  of  the  bacterial 
mixture  and,  while  still  liquid,  are  laid  in  an  almost  horizontal  position 
upon  a  block  of  ice,  which  has  been  grooved  slightly  by  means  of  a  test 
tube  filled  with  hot  water.  The  test  tube  containing  the  gelatin,  after  being 
placed  in  this  groove,  is  rapidly  revolved  by  passing  the  fingers  of  the  right 
hand  across  it,  while  its  base  is  carefully  steadied  with  the  left  hand.  If 
the  revolving  is  carried  out  with  sufficient  speed,  the  gelatin  will  harden 
in  a  thin  layer  on  the  inner  surface  of  the  tube.  The  colonies  will  develop 
in  this  layer  and  may  be  "fished"  by  means  of  a  platinum  wire  with  bent 
point  introduced  into  the  tube.  This  method  is  useful  for  certain  purposes, 
but  is  too  inconvenient  for  routine  work.  It  is  now  rarely  used. 


1  Esmarch,  Zeit.  f.  Hyg.,  i,  1886. 


178  BIOLOGY  AND  TECHNIQUE 

Separation  of  Bacteria  by  Surface  Streaking. — When  it  is  neces- 
sary to  isolate  bacteria  like  the  gonococcus  Bacillus  influenzas,  the 
pneumococcus,  and  others,  which,  because  of  great  sensitiveness  to 
environment  and  possibly  a  preference  for  free  oxygen,  are  not 
readily  grown  in  pour  plates,  it  is  often  advantageous  first  to  pour 
plates  of  suitable  media,  allow  them  to  harden,  and  then  gently 
smear  over  their  surfaces  dilutions  of  the  infectious  material,  usually 
in  three  or  four  parallel  streaks. 

Upon  such  plates,  if  dilutions  have  been  properly  made,  and 
this  is  only  a  question  of  judgment  based  upon  an  estimation  of  the 
numbers  of  bacteria  in  the  original  material,  discrete  colonies  of 
the  microorganisms  sought  for  may  develop,  and  can  be  " fished" 
in  -the  usual  manner. 

The  media  most  favorable  for  the  cultivation  of  various  micro- 
organisms will  be  discussed  in  the  sections  dealing  with  the  in- 
dividual species. 

BARBER  PIPETTE  METHOD  FOR  THE  ISOLATION  OF  SINGLE  MICRO- 
ORGANISMS.— Although  we  consider  this  one  of  the  very  important 
methods  of  bacteriological  study,  we  shall  give  no  extensive  descrip- 
tion of"  the  apparatus  or  the  manner  of  using  it,  because  both  are 
too  complicated  to  permit  of  satisfactory  use  from  text  book  descrip- 
tion. The  principle  of  the  method  depends  upon  a  specially  pre- 
pared mechanical  stage  adjusted  to  a  compound  microscope  on  which 
there  is  a  moist  chamber  closed  with  a  large  coverglass,  on  the 
bottom  of  which  drops  of  fluid,  containing  bacteria  can  be  placed. 
A  very  fine  glass  pipette,  manipulated  by  a  specially  constructed 
pipette  holder  and  with  a  rubber  tubing  attached,  is  governed  by 
observation  through  the  microscope  and  by  means  of  it  small  drops 
of  the  fluid  are  taken  up,  an  attempt  being  made  to  obtain  a  single 
microorganism  in  these  drops.  With  a  little  practice  this  can  be 
accomplished,  and  these  separate  drops  in  which  the  individual 
bacteria  can  be  seen  swimming  about,  are  made  to  suspend  from 
the  bottom  of  the  cover,  slip,  closing  the  top  of  the  moist  chamber. 
The  apparatus  can  be  understood  and  worked  only  by  practice  and 
suitable  instruction,  together  with  a  study  of  the  description  given 
by  Barber  in  his  article  in  the  Philippine  Journal  of  Science.2  The 
apparatus  is  made  by  a  number  of  firms  under  the  name  of  the 


2  Barber,  Philippine  Jour,  of  Science,  Sec.  B,  Vol.  9,  No.  4,  August,  1914. 


METHODS   USED   IN   CULTIVATION  OF   BACTERIA  179 

Barber  Single  Cell  Apparatus,  and  no  description  that  could  be  given 
in  a  text  book  would  be  of  sufficient  value  to  be  allowed  to  take 
up  space. 

ANAEROBIC    METHODS 

We  have  seen  in  a  preceding  chapter  (p.  28)  that  many  bacteria, 
the  so-called  anaerobes,  will  develop  only  in  an  environment  from 
which  free  oxygen  has  been  excluded. 


FIG.  15.— DEEP  STAB 
CULTIVATION  OF  AN- 
AEROBIC BACTERIA. 


FIG.  16. — DEEP  STAB 
CULTIVATION  OF  AN- 
AEROBIC BACTERIA. 


The  exclusion  of  oxygen  for  purposes  of  anaerobic  cultivation 
may  be  accomplished  by  a  variety  of  methods,  depending  upon  a 
few  simple  principles  which  have  been  applied,  either  separately 
or  in  combination,  by  many  workers. 

The  earliest  methods  depended  upon  the  simple  exclusion  of  air 
by  mechanical  devices.  In  other  methods,  the  oxygen  of  the  air 
is  displaced  by  inert  gases  (hydrogen),  and  others  again  depend 


180  BIOLOGY  AND  TECHNIQUE 

upon  the  oxygen-absorbing  qualities  of  alkaline  solutions  of  pyro- 
gallol. 

Cultivation  by  the  Mechanical  Exclusion  of  Air. — Koch  succeeded  in 
growing  anaerobic  bacteria  upon  plates  by  simply  dropping  upon  the  surface 
of  the  inoculated  agar  or  gelatin  a  flat  piece  of  sterile  mica.  This  method, 
however,  rarely  succeeds  in  sufficiently  excluding  the  air. 

LIBORIUS'  METHOD.3 — This  method  consists  in  the  use  of  deeply  filled 
tubes  of  agar  or  gelatin,  from  which  all  oxygen  has  been  removed  by  boiling 
for  fifteen  minutes  or  more.  It  is  advantageous,  as  has  been  pointed  out 
in  the  section  on  anaerobiosis,  that  media  used  for  this  purpose  should 
contain  carbohydrates  in  some  form,  preferably  glucose.  After  boiling,  the 
tubes  are  rapidly  transferred  to  ice  water  so  that  as  little  oxygen  as  possible 
may  be  absorbed  during  the  hardening  of  the  medium.  The  tubes  are  then 
inoculated  by  deep  stabs.  After  inoculation,  the  medium  may  be  covered 
with  a  thin  layer  of  agar,  gelatin,  or  oil  (albolin),  and  further  sealed  with 
sealing-wax  to  prevent  oxygen-absorption. 

This  method  may  be  utilized  for  the  isolation  of  anaerobes  (as  in  the 
original  method  of  Liborius)  by  inoculating  the  medium  just  before  it 
solidifies.  The  tubes  may  be  gently  shaken  in  order  to  distribute  the  bacteria 
throughout  the  medium  and  then  rapidly  cooled.  In  this  case  colonies  which 
develop  may  be  scattered  throughout  the  deeper  layers  of  the  agar  or  gelatin, 
and  may  be  "fished"  after  breaking  the  tube. 

ESM ARCH'S  METHOD.* — Von  Esmarch  has  applied  the  principles  of  his 
roll-tube  to  the  cultivation  of  anaerobic  bacteria.  Gelatin  tubes  are  inoculated 
as  above  and  roll-tubes  prepared.  The  tubes  are  then  set  into  cold  water 
to  prevent  melting  of  the  thin  gelatin  layer  and  the  interior  of  the  tube 
is  filled  with  melted  gelatin. 

Roux's  METHOD.5 — Anaerobic  bacteria  are  cultivated  by  sucking  the  inocu- 
lated gelatin  or  agar  into  narrow  tubes,  which  are  then  closed  at  both  ends 
by  fusing  in  the  flame.  After  growth  has  taken  place  the  tubes  are  broken 
and  the  organism  recovered  by  "fishing." 

FLUID  MEDIA  COVERED  WITH  OIL. — Erlenmeyer  flasks  or  other  vessels  are 
partially  filled  with  glucose-bouillon  over  which  a  thin  layer  of  albolin  or 
other  oil  is  allowed  to  flow.  The  oxygen  is  driven  out  of  the  liquid  by 
vigorous  boiling  for  fifteen  minutes  or  more. 

It  should  be  remembered  whenever  using  this  or  similar  methods  that 
a  layer  of  fluid  oil  does  not  form  an  impermeable  seal.  By  covering  an 
alkaline  pyrogallol  solution  with  oil  it  can  easily  be  shown  that  oxygen 


'Liborius,  Zeit.  f.  Hyg.,  i,   1886. 
4  Von  Esmarch,  loc.  cit. 
«Koux,  Ann.  Past.,  i,  1887. 


METHODS   USED   IN   CULTIVATION  OF  BACTERIA 


181 


slowly  diffuses  through  the  oil  into  the  medium  below.  In  using  paraffin 
oil  on  anaerobic  cultures  it  must  be  remembered  that  liquid  oil  is  a  very 
incomplete  seal  and  that  solid  paraffin  or  any  other  solid  seal  is  much  more 
efficient. 

The  simple  exclusion  of  air,  also,  is  the  principle  underlying  the  cultiva- 
tion of  anaerobic  bacteria  in  the  closed  arm  of  a  Smith  fermentation  tube. 

WRIGHT'S  METHOD.6 — Wright  has  described  a  simple  and  excellent  method 
for  the  cultivation  of  anaerobic  bacteria  in  fluid 
media.  The  apparatus  necessary  is  easily  im- 
provised with  the  materials  at  hand  in  any 
laboratory.  A  short  piece  of  glass  tubing,  con- 
stricted at  both  ends  and  fitted  at  each  end  with 
a  small  piece  of  soft-rubber  tubing,  is  inserted 
into  a  test  tube  containing  nutrient  broth.  The 
upper  end  of  the  inserted  glass  tubing  is  con- 
nected by  the  rubber  with  a  pipette  passed 
through  the  cotton  plug  in  the  tube.  The  entire 
apparatus,  plus  broth,  may  be  sterilized  after 
being  put  together.  When  a  transplant  is  made, 
the  fluid  in  the  test  tube  is  inoculated  as  usual. 
The  fluid  is  then  sucked  up  into  the  glass  tubing 
until  this  is  completely  filled.  A  downflow  of 
the  fluid  is  then  prevented  by  placing  the  finger 
over  the  pipette  through  which  the  suction  has 
been  made  or  by  constricting  a  small  piece  of 
rubber  tubing  attached  to  the  upper  end  of  the 
pipette.  The  entire  system  of  tubes  is  then 
pushed  downward  in  such  a  way  that  both  pieces 
of  rubber  tubing,  attached  to  the  ends  of  the 
little  glass  chamber,  are  kinked.  The  entire 
apparatus  may  then  be  incubated.  Growth  of 
anaerobic  bacteria  takes  place  within  the  air- 
tight chamber  formed  by  the  short  glass  tubing 
within  the  test  tube.  The  fluid  in  the  test  tube, 
outside  of  this  chamber,  usually  remains  clear. 

When  cultivation  has  been  successful,  the 
bacteria  may  be  obtained  either  for  morphological 
study  or  for  further  cultivation,  by  simply  allowing  the  fluid  to  flow  out 
of  the  little  air-tight  chamber  back  into  the  test  tube.  The  method  is 
simple  and  usually  successful. 


FIG.  17. — WRIGHT'S  METHOD 
OF  ANAEROBIC  CULTIVATION 
IN  FLUID  MEDIA. 


6  Wright,  J.  II.     Quoted  from  Mallory  and  Wright,  "Path.  Technique/'  Phila., 
1904. 


182 


BIOLOGY  AND  TECHNIQUE 


Methods  Based  upon  the  Displacement  of  Air  by  Hydrogen.— The  prin- 
ciple of  air-displacement  by  hydrogen,  first  utilized  by  Hauser,7  has  been 
widely  applied  to  the  cultivation  of  anaerobic  bacteria.  In  substance  it 
consists  of  the  conduction  of  a  stream  of  hydrogen  through  an  air-tight 
chamber  in  which  plates  or  tubes  containing  inoculated  media  have  been 
placed. 

For  the  production  of  hydrogen,  the  most  convenient  apparatus  is  the 
Kipp  hydrogen  generated  from  zinc  and  sulphuric  acid  and  this  may  be 


FIG.  18. — NOVY  JAR. 

passed  through  a  series  of  Woulfe-bottles,  containing  solutions  of  lead 
acetate  and  of  pyrogallic  acid,  to  remove  traces  of  sulphuretted  hydrogen 
and  of  oxygen,  respectively,  of  Lugol's  solution  to  absorb  traces  of  acid 
vapor,  and  of  one  with  a  silver-nitrate  solution  to  take  up  any  hydrogen 
arsenide. 

For  the  preparation  of  anaerobic  conditions  where  very  rigid  anaerobiosis 
is  necessary,  nitrogen  may  be  used,  which  can  be  bought  in  tanks  from  com- 
mercial firms. 


Hauser,  ( i  Ueber  Faulnissbakterien, ' '  1885. 


METHODS   USED   IN   CULTIVATION  OF  BACTERIA  183 

For  anaerobic  cultivation  upon  solid  media,  the  inoculated  tubes  or  plates 
are  placed  in  an  apparatus  such  as  the  Novy  jar.  This  is  connected  with 
the  hydrogen  apparatus  and  hydrogen  allowed  to  flow  through  it  for  five 
or  ten  minutes,  and  the  stop-cocks  then  closed. 

In  applying  the  hydrogen  method  to  fluid  media,  flasks  containing  the 
broth  are  fitted  with  sterile,  tightly  fitting  rubber  stoppers  perforated  by 
two  holes,  through  which  glass  tubes  are  passed.  One  of  these  tubes,  the 
inlet,  passes  below  the  surface  of  the  liquid.  The  other  one,  the  outlet, 
extends  only  a  short  distance  below  the  stopper  and  is  always  kept  above 
the  surface  of  the  medium.  The  flasks  are  inoculated  and  hydrogen  is 
passed  through  the  medium  so  that  it  enters  the  long  tube,  bubbles  up  through 
the  fluid,  and  leaves  by  the  short  tube.  The  broth  may  be  covered  with  a 
thin  layer  of  liquid  paraffin  or  albolin. 

The  Use  of  Pyrogallic  Acid  Dissolved  in  Alkaline  Solutions  for  Oxygen 
Absorption. — Buchner8  has  applied  the  principle  of  chemical  absorption  for 
the  removal  of  oxygen  to  the  cultivation  of  anaerobic  bacteria.  This  has 
been  made  use  of  in  a  number  of  different  ways.  The  method  is  based  upon 
the  fact  that  alkaline  solutions  of  pyrogallol  possess  the  power  of  absorbing 
large  quantities  of  free  oxygen.  At  first  such  solutions  are  of  a  light 
straw-color,  which  becomes  dark  brown  as  oxygen  is  absorbed.  The  absorp- 
tion of  all  the  oxygen  in  the  environment  may  be  assumed  when  there  is  no 
further  deepening  of  the  brown  color. 

Buchner  first  utilized  this  principle  by  placing  a  small  wire  or  glass  holder 
within  a  large  test  tube,  dropping  dry  pyrogallol  (pyrogallic  acid)  into  the 
bottom  of  this  tube,  then  running  thirty  per  cent  sodium  hydrate  solution 
into  it,  and  inserting  within  this  large  tube  a  smaller  test  tube  containing 
the  inoculated  culture  medium.  The  large  tube  was  then  tightly  closed 
with  a  rubber  stopper.  In  this  way,  the  air  space  surrounding  the  smaller 
tube  was  rendered  oxygen  free.  An  excellent  little  trick  with  which  to 
employ  the  Buchner  tube  method  is  to  pack  lightly  over  the  dry  pyrogallic 
acid  in  the  bottle  a  small  piece  of  absorbent  cotton.  This  prevents  the 
immediate  solution  of  the  pyrogallic  acid,  and  allows  one  time  to  pour  in 
the  KOH  solution,  insert  the  smaller  tube  inside  the  Buchner  tube,  and 
tightly  insert  the  rubber  stopper  in  place  before  solution  and  oxygen  absorp- 
tion has  begun. 

A  simple  modification  of  the  preceding  method  of  Buchner  has  been 
devised  by  Wright.9  Stab-cultures  of  gelatin  or  agar  in  test  tubes  are  made 
in  the  usual  way.  The  cotton  stopper  closing  the  tube  is  then  thrust  into 
the  tube  to  such  a  depth  that  its  upper  end  lies  at  least  1  cm.  below  the 
mouth  of  the  tube.  A  small  quantity  of  sodium  or  potassium  hydrate  solution 


*  Buchner,  Cent.  f.  Bakt.,  I,  iv,  1888. 

9  Wright,  Jour,  of  the  Boston  Soc.  of  Med.  Sci.,  Dec.,  1900. 


184 


BIOLOGY  AND  TECHNIQUE 


in  which  some  pyrogallic  acid  has  been  dissolved,  is  then  allowed  to  flow 
on  to  the  cotton  of  the  plug  and  the  mouth  of  the  tube  is  immediately  sealed 
by  a  tightly  fitting  rubber  stopper.  The  cotton  stopper  in  these  cases  must 
be  made  of  absorbent  cotton;  1.5  to  2.5  c.c.  of  the  pyrogallic  acid  solution 
is  usually  sufficient  for  test  tubes  of  ordinary  size. 

For  cultivation  of  anaerobic  bacteria  upon  agar  slants,  a  simple  technique 


FIG.  19. — WEIGHT'S  METH- 
OD OF  ANAEROBIC  CULTIVA- 
TION BY  THE  USE  OF  PYRO- 

GALLIC  ACID  SOLUTION. 


FIG.  20. — JAR  FOR  ANAEROBIC  CUL- 
TIVATION. 


may  be  applied  and  easily  improvised  in  the  laboratory  as  follows:  the 
tube  of  slant  agar  is  inoculated  with  the  infectious  material  in  the  usual 
way.  It  is  then,  with  stopper  removed,  inverted  into  a  tumbler  or  beaker 
containing  about  a  gram  of  dry  pyrogallic  acid.  A  small  quantity  of  a 
five  per  cent  or  three  per  cent  sodium  hydrate  solution  is  then  run  into  the 
tumbler  and  this  is  covered  with  a  thin  layer  of  liquid  paraffin  or  albolin 
before  the  pyrogallic  acid  has  been  completely  dissolved.  In  this  way,  com- 


METHODS  USED   IN   CULTIVATION  OF  BACTERIA 


185 


pletely  anaerobic  conditions  are  obtained  in  the  tube  and  the  growth  of 
anaerobes  takes  place  upon  the  surface  of  the  slant. 

For  the  cultivation  of  anaerobes  in  Petri  dishes,  for  purposes  of  separa- 
tion, a  combination  of  the  pyrogallic  acid  method  and  the  hydrogen  displace- 
ment methods  is  often  employed.  For  this  purpose  the  jars  devised  by 
Novy  and  by  Bulloch  are  extremely  convenient. 

In  using  the  Novy  jar,  the  inoculated  plates  are  set  upon  a  wire  frame 
resting  about  an  inch  above  the  bottom  of  the  jar.  The  cover  is  then  tightly 
set  in  place  and  the  air  in  the  jar  exhausted  by  means  of  a  suction  pump. 
The  arrangement  of  the  double  stop-cock  in  the  top  renders  it  possible  now, 
by  simply  turning  this,  to  admit  hydrogen  from  a  Kipp  generator  into  the 
jar.  The  process  of  alternate  exhaustion  and  admission  of  hydrogen  may 
be  several  times  repeated. 


FIG.  21. — APPARATUS  FOR  COMBINING  THE  METHODS  OF  EXHAUSTION,  HYDROGEN 
REPLACEMENT  AND  OXYGEN  ABSORPTION. 

A  combination  of  air  exhaustion,  oxygen  absorption,  and  hydrogen  replace- 
ment may  be  practiced  in  jars  such  as  that  shown  in  Fig.  21.  Dry  pyrogallic 
acid  is  placed  in  the  bottom  of  the  jar  and  the  cover  tightly  fitted.  An 
opening  in  the  side  of  the  jar  connects  its  interior  with  a  bottle  containing 
sodium  or  potassium  hydrate  solution.  Through  the  stopper  of  this  bottle 
pass  two  glass  tubes,  one  of  them  of  such  length  that  it  can  be  pushed 
down  into  the  alkaline  solution,  or  pulled  upward  above  the  level  of  the 
fluid.  By  this  means  the  KOH  can  be  sucked  into  the  jar  after  a  vacuum 
has  been  produced  with  the  exhaustion  pump.  The  other  glass  tube  is 
short,  passing  just  through  the  stopper  and  at  the  top  made  in  the  form 
of  a  T,  one  arm  of  the  T  being  connected  with  a  Kipp  hydrogen  generator, 
the  other  with  a  suction-pump. 

A  simple  method  for  the  separation  of  anaerobes  in  plates  without  the 
use  of  hydrogen  or  of  specially  constructed  jars,  may  be  carried  out  as 
follows :  10  The  apparatus  used  consists  of  two  circular  glass  dishes,  fitting 

™  Zinsser,  Jour.  Exp.  Med.,  viii,  1906. 


186  BIOLOGY  AND  TECHNIQUE 

one  into  the  other  as  do  the  halves  of  a  Petri  dish,  and  similar  to  these 
in  every  respect  except  that  they  are  higher,  and  that  a  slightly  greater 
space  is  left  between  their  sides  when  they  are  placed  together.  The  dishes 
should  be  about  three-fourths  to  one  inch  in  height,  they  need  be  of  no 
particular  diameter,  although  those  of  about  the  same  size  as  the  usual 
Petri  dish  are  most  convenient.  An  important  requirement  necessary  for 
the  success  of  the  method  is  that  the  trough  left  between  the  two  plates, 
when  put  together,  shall  not  be  too  broad,  a  quarter  of  an  inch  being  the 
most  favorable. 

Into  the  smaller  of  these  plates  the  inoculated  agar  is  poured  exactly 
as  into  a  Petri  dish  in  the  ordinary  aerobic  work.  Prolonged  boiling 
of  the  agar  before  plating  is  not  essential.  When  the  agar  film  has 
become  sufficiently  hard  on  the  bottom  of  the  smaller  dish,  the  entire  ap- 
paratus is  inverted.  The  smaller  dish  is  now  lifted  out  of  the  larger,  and 
placed,  still  inverted,  over  a  moist  surface — a  towel  or  the  wet  surface  of 
the  table — to  prevent  contamination.  Into  the  bottom  of  the  larger  dish, 
which  now  stands  open,  there  is  placed  a  quantity  (about  3  grams)  of  dry 
pyrogallic  acid.  Into  this,  over  the  pyrogallic  acid,  the  smaller  dish,  still 
inverted,  is  then  placed.  A  five  per  cent  solution  of  sodium  hydrate  is 
poured  into  the  space  left  between  the  sides  of  the  two  dishes,  in  quantity 
sufficient  to  fill  the  receiving  dish  one-half  full.  While  this  is  gradually 
dissolving  the  pyrogallic  acid,  albolin,  or  any  other  oil  (and  this  is  the  only 
step  that  requires  speed),  is  dropped  from  a  pipette,  previously  filled  and 
placed  in  readiness,  into  the  same  space,  thus  completely  sealing  the  chamber 
formed  by  the  two  dishes. 

If  these  steps  have  been  performed  successfully,  the  pyrogallic  solution 
will  at  this  time  appear  of  a  light  brown  color,  and  the  smaller  plate,  with 
its  agar  film,  will  float  unsteadily  above  the  other.  Very  rapidly,  as  the 
pyrogallic  acid  absorbs  the  free  oxygen  in  the  chamber,  this  plate  is  drawn 
down  close  to  the  other,  and  the  acid  assumes  a  darker  hue,  which  remains 
without  further  deepening  even  after  three  or  four  days'  incubation. 

We  have  described  a  considerable  number  of  methods  of  anae- 
robic cultivation  which  have  been  in  use.  Following  our  general 
purpose,  however,  of  emphasizing  the  methods  that  we  ourselves 
have  found  most  useful,  we  will  describe  in  the  following  paragraphs 
the  methods  which  we  are  using  as  routine  for  anaerobic  work  in 
our  own  laboratory,  and  which  we  believe  are  the  most  practical. 

For  anaerobic  cultivation  on  agar  slants,  we  use  the  Buchner 
tube  method  as  described  above. 

For  spirochaete  cultivation,  etc.,  we  use  the  Noguchi  method 
of  narrow  deep  agar  or  broth  tubes  with  tissue  at  the  bottom,  sterile 


METHODS  USED  IN  CULTIVATION  OF  BACTERIA  187 

oil  at  the  top,  placed  into  anaerobic  jars,  or  incubated  without  jars 
according  to  purpose. 

For  anaerobic  jar  methods,  we  hardly  ever  use  hydrogen,  em- 
ploying one  of  the  two  following : 

Jars  may  be  of  any  size  or  shape,  provided  they  are  well  constructed 
strong  museum  jars  with  well  fitting  lids,  preferably  ground  glass  surfaces 
on  lid  and  top  of  jar,  and  with  one  perforation  in  the  top.  Stoppers  and 
glass  tubing  are  fitted  into  the  top  of  the  jars  with  Major's  cement,  and 
recemented  every  time  the  jars  are  used.  Glass  tubing  which  communicates 
with  the  exterior  through  the  cemented  joints  is  drawn  out  in  the  flame, 
along  its  course  connecting  with  the  suction  pump,  and  closure  is  effected 
by  sealing  in  these  narrow  places  with  the  flame. 

The  fitting  of  the  lid  for  closure  is  accomplished  by  a  thick  layer  of 
sculptors'  plastocene,  a  smooth  layer  of  which  is  placed  on  the  top  of  the 
jar,  and  another  on  the  lips  of  the  lid  before  the  lid  is  put  in  place.  It  is 
useful  to  have  a  vacuum  gauge  cemented  into  the  lid  so  that  the  functioning 
of  the  suction  pump  can  be  controlled.  This,  however,  is  not  absolutely 
necessary.  A  small  tube  containing  a  weak  methylene  blue  solution  in  1  per 
cent  glucose  broth  is  placed  inside  the  jar  with  the  cultures.  Decolorization 
of  this  on  the  following  morning  will  prove  anaerobiosis. 

I.  One  method  which  can  be  used  for  Petri  plates  or  tubes    consists  in 
placing  a  suitable  amount  of  dry  pyrogallic  acid  into  the  bottom  of  the 
jar.     The  cultures  are  then  inserted  and  next  to  them  is  placed  an  envelope 
of  thick  brown  paper,  such  as  used  by  commercial  houses,  which  is  torn  so 
as  to  be  open  at  the  top  and  to  form  a  sort  of  elongated  bag  or  cornucopia. 
The  glass  top  of  the  lid  is  then  connected  with  the  suction  pump  before 
it  is  placed  on  the  jar,  the  suction  pump  started  while  an  assistant  holds 
the  lid  ready  for  placement  on  the  jar.     A  suitable  amount  of  20  per  cent 
KOH   is   then   poured   into   the   envelope   and   the   lid   immediately   placed 
in  position  on  the  top  of  the  jar,  pushed  down  on  all  sides  so  that  the 
plastocene  flattens  out  between  the  lid  and  the  jar.    The  suction  immediately 
begins  to  draw  the  air  out  of  the  jar,  before  the  KOH  solution  works  its 
way  through  the  envelope  and  begins  to  dissolve  the  pyrogallol.     The  suction 
is  then  continued  for  a  suitable  length  of  time,  and  while  the  pump  is  still 
going  a  Bunsen  burner  is  placed  under  a  narrow  place  in  the  connection 
glass  tube  and  this  is  sealed  in  the  flame.    This  method  if  successfully  carried 
out,  gives  almost  complete  anaerobiosis. 

II.  The  second  method  is  that  of  Mclntosh  and  Fildes,11  which 
is,  in  our  opinion,  the  best  anaerobic  method  for  application  to  the 

"Mclntosh  and  Fildes,  Lancet,  190,  1916,  768.  Methylene  blue  added  in 
sufficient  quantity  to  10  c.c.  of  a  2  per  cent  dextrose  alkaline  broth  to  give  a 
distinct  blue  color,  is  the  most  convenient  anaerobic  indicator.  It  depends  on 


188  BIOLOGY  AND  TECHNIQUE 

growth  of  tubes  and  plates  in  anaerobic  jars.  It  depends  upon  the 
removal  of  oxygen  by  the  oxidation  of  hydrogen  under  the  influence 
of  palladinized  asbestos  wool.  Pfuhl  had  previously  used  the 
catalytic  action  of  platinum  sponge  for  anaerobic  cultivation  in 
broth  tubes.  Mclntosh  and  Fildes  adopted  and  improved  upon  this 
method.  The  principle  of  the  method  is  to  suspend  in  an  air  tight 
jar  a  bit  of  asbestos  wool  impregnated  with  platinum  or,  better, 
with  palladium  black.  Hydrogen  is  then  allowed  to  pass  in  until 
no  oxygen  remains.  We  have  used  this  method  in  our  own  labora- 
tory with  great  success  where  a  number  of  the  workers  compared 
it  with  other  methods,  and  it  has  been  used  successfully  at  the 
Rockefeller  Institute  by  Olitsky  and  others. 

An  ordinary  museum  jar,  such  as  those  used  for  anaerobic  work,  is  the 
vessel  employed.  The  lid  is  perforated  for  the  passage  of  hydrogen,  and 
from  the  bottom  of  the  lid  there  is  suspended  a  bit  of  the  impregnated 
asbestos  wool,  inclosed  in  a  small  cage  of  copper  wire.  About  0.25  gram 
of  the  asbestos  wool  is  the  amount  recommended  by  Mclntosh  and  Fildes. 

"The  palladium  asbestos  (about  40  per  cent)  is  made  by  weighing  out 
0.25  gram  of  asbestos  wool,  placing  it  in  a  small  evaporating  dish,  and 
adding  1.5  c.c.  of  a  10  per  cent  solution  of  palladium  chlorid.  The  wool 
is  then  molded  into  a  flat  mass  about  one  inch  square,  and  the  dish  gently 
heated  until  the  wool  is  dry.  Since  the  palladium  chlorid  is  difficult  to 
dissolve,  the  addition  of  a  little  concentrated  hydrochloric  acid  may  be  neces- 
sary. The  palladium  chlorid  is  then  reduced  by  heating  the  impregnated 
wool,  first  in  a  smoky  gas  flame  until  it  is  coated  with  a  layer  of  carbon, 
and  then  in  a  blow-pipe.  The  palladium  asbestos  should  now  be  able  to 
light  a  jet  of  hydrogen  which  is  made  to  impinge  on  it." 

For  ordinary  purposes  the  palladium  asbestos  can  be  bought.  For  closure 
of  lids  upon  the  jars,  we  have  used  throughout  plastocene,  the  material  used 
by  sculptors  for  molding. 

In  using  the  jars  the  culture  tubes  are  placed  inside  the  jars  and  the 
lid  rim  and  the  jar  rim  are  covered  with  plastocene.  The  copper  gauze, 
with  the  asbestos  wool,  is  detached  and  held  over  a  flame  until  red  hot,  and 
is  then  rapidly  put  in  place,  closing  the  jar.  Through  the  perforation  with 
proper  connections  made  (best  a  glass  stopper  connection)  hydrogen  is 
allowed  to  flow  in  very  slowly,  best  from  a  liquid  hydrogen  cylinder,  with 
careful  regulating  check  valve.  It  is  very  necessary  to  take  careful 

the  fact  that  in  the  absence  of  oxygen  the  reducing  action  of  an  alkaline  solution 
of  dextrose  changes  the  methylene  blue  to  the  colorless  luecomethylene  blue.  On 
exposure  to  the  oxygen  of  the  air,  the  leucomethylene  blue  is  oxidized  back  to 
the  colored  compound. 


METHODS  USED  IN  CULTIVATION  OF  BACTERIA  189 

precautions  that  the  hydrogen  is  not  allowed  to  enter  too  fast,  or 
else  explosion  may  follow,  and  it  is  best  to  make  some  arrange- 
ment like  that  used  in  our  laboratory  by  Dr.  Teague  where  the 
hydrogen  is  first  let  into  a  glass  chamber  over  water,  and  allowed  to  flow 
in  very  gradually  by  graded  pressure.  The  main  thing  is  to  so  work  the 
apparatus  that  a  very  slow  jet  of  hydrogen  is  made  to  flow  in  close  to  the 
palla-dmized  asbestos,  while  this  is  still  hot.  A  small  film  of  water  will 
begin  to  form  on  the  sides  of  the  tube,  and  this  is  continued  until  a  negative 
pressure  has  distinctly  developed  in  the  jar.  Judgment  concerning  the 
amount  of  hydrogen  that  should  be .  let  in  can  be  attained  with  practice. 
Closure  of  the  jar  may  be  then  made  more  firm  with  paraffin  or  any  of 
the  other  ordinary  methods  of  making  such  jars  air  tight.  Plastocene  in 
sufficient  amounts  has  usually  served  our  purpose. 

We  do  not  illustrate  this  method  because  illustrations  rarely  help  an 
experienced  bacteriologist  to  any  great  degree,  and  it  is  best  to  see  the 
apparatus  work  in  some  laboratory  where  it  is  in  use.  Many  modifications 
are  possible,  but  the  principle  is  easy  to  apply  in  a  great  many  different 
ways. 

By  this  method  it  is  possible  to  decolorize  methylene  blue  tubes  put  into 
the  jars,  completely  over  night,  and  we  believe  that  this  method  properly 
applied  gives  a  practically  perfect  anaerobiosis. 

The  Use  of  Fresh  Sterile  Tissues  as  an  Aid  to  Anaerobic  Cul- 
tivation.— The  addition  of  small  pieces  of  fresh  sterile  tissue  (rabbit 
or  guinea-pig)  to  culture  tubes,  either  solid  or  fluid,  greatly  favors 
the  growth  of  anaerobic  bacteria.  By  such  a  method  anaerobes  can 
be  made  to  develop  even  when  other  precautions  for  the  establish- 
ment of  anaerobiosis  are  imperfectly  observed.  This  was  noticed 
first  by  Theobald  Smith  and  by  Tarozzi  and  has  become  an  extremely 
useful  reenforcement  to  other  methods.  It  has  been  utilized  most 
extensively  by  Noguchi  of  recent  years  in  his  technique  for  the 
cultivation  of  various  treponemata.  The  simplest  way  to  apply 
this  method  is  to  place  a  piece  of  freshly  excised  rabbit  kidney, 
testicle,  or  spleen  into  the  bottom  of  a  high  test  tube  (20  cm.)  and 
then  pouring  over  it  the  culture  fluid.  Kidney  or  other  tissues  are 
more  suitable  for  this  purpose  than  liver  tissue  since  the  latter  is 
not  easily  obtained  in  a  sterile  condition,  bacteria  often  getting  into 
it  during  life  through  the  portal  circulation.  The  action  of  the 
tissues  depends  upon  its  great  reducing  power. 

PARTIAL  OXYGEN  TENSION  FOR  THE  GROWTH  OF  BACTERIA. — In  1916 
Wherry  and  Oliver12  found  that  partial  anaerobiosis  was  favorable 

12  Wherry  and  Oliver,  Lancet-Clinic,  115,  1916,  306. 


190  BIOLOGY  AND  TECHNIQUE 

for  the  growth  of  gonococcus.  Cohen  and  Markle13  applied  this  to 
meningococcus  and  found  again  that  partial  anaerobiosis  favored 
growth.  It  was  later  found  by  Wherry  and  Erwin,14  as  well  as  by 
Cohen  and  Fleming15  that  the  growth  of  a  number  of  different 
bacteria  was  favored  when  the  air  was  partially  replaced  by  carbon 
dioxide.  Gates16  confirmed  this,  as  did  Kohman.17  We  have  tried 
this  out  in  our  own  laboratory  and,  like  other  observers,  have  found 
that,  in  the  case  of  meningococcus  and  also  with  the  influenza 
bacillus,  growth  is  definitely  stimulated  by  replacing  about  10  per 
cent  of  the  air  with  carbon  dioxide'.  Both  Kohman,  Gates  and  others 
believe  that  the  principle  underlying  this  is  due  to  the  effect  of  the 
carbon  dioxide  on  the  reaction  of  the  medium.  As  the  organisms 
grow  they  produce  acid  which  displaces  equivalent  amounts  of  dis- 
solved carbon  dioxide,  in  consequence  of  which  the  acidity  is  hot 
materially  increased. 

THE  INCUBATION  OF  CULTURES 

After  bacteria  have  been  transferred  to  suitable  culture  media, 
it  is  necessary  to  expose  them  to  a  temperature  favorable  to  their 
development.  In  the  case  of  many  saprophytes,  the  ordinary  room 
temperature  is  sufficiently  near  the  optimum  to  obviate  the  use  of 
any  special  apparatus  for  maintaining  a  suitable  temperature;  in 
the  case  of  most  pathogenic  bacteria,  however,  the  body  temperature 
of  man,  37.5°  C.,  is  either  a  necessary  requirement  for  their  growth, 
or  at  any  rate  favors  speedy  and  characteristic  development.  . 

For  the  purpose  of  obtaining  a  uniform  temperature  of  any 
required  degree,  the  apparatus  in  general  use  is  the  so-called  incu- 
bator or  thermostat.  This  may  be  adjusted  for  gelatin  cultivation 
at  20  to  22°  C.,  or  for  agar,  broth,  or  other  media  at  37.5°  C. 

Incubators,  while  varying  in  detail,  are  all  constructed  upon  the 
same  principles.  They  consist  of  double-walled  copper  chambers, 
which  are  fitted  with  a  set  of  double  doors,  the  outer  being  made 
of  asbestos-covered  metal,  the  inner  of  glass.  The  space  between 
the  two  walls  is  filled  with  water,  which,  being  a  poor  heat  con- 

13  Cohen  and  Markle,  Abst.  of  Bacter.,  2,  1918,  10. 

14  Wherry  and  Erwin,  Jour,  of  Infec.  Dis.,  22,  1918,  194. 

15  Cohen  and  Fleming,  Jour,  of  Infec.  Dis.,  23,  337,  1918, 
19  Gates,  Jour,  of  Exper.  Med.,  29,  325,  1919. 

"  Kohman,  Jour,  of  Bacter.,  4,  1919. 


METHODS   USED   IN   CULTIVATION   OF   BACTERIA 


191 


ductor,  tends  to  prevent  rapid  changes  of  temperature  within  the 
chamber  as  the  result  of  changes  in  the  external  environment.  Both 
walls  are  perforated  above  by  openings  to  admit  thermometers  into 
the  interior  and  one  wall  is  perforated  so  that  a  thermo-regulator 
may  be  inserted  into  the  water  jacket.  The  under  surface  of  the 
chamber  is  heated  by  a  gas  flame,  the  size  of  which  is  automatically 
regulated  by  the  thermo-regulator. 

A  number  of  thermo-regulators  are  on  the  market,  all  of  them  con- 
structed upon  modifications  of  the  same  principle.  One  of  the  most  efficient 
of  those  in  common  use  is  that  shown  in  Fig.  22.  This  consists  of  a  long 
tube  of  glass  fitted  with  a  metal  cap  through  which  an  inlet  tube  (A)  projects 


n 

i. 


FIGS.  22,  23. — THERMO  REGULATOR. 

into  the  interior.  Slightly  below  the  middle  of  the  tube  there  is  a  glass 
diaphragm  separating  its  interior  into  two  compartments.  In  the  middle 
of  the  diaphragm  an  aperture  leads  into  a  spiral  of  glass  which  projects 
into  the  lower  compartment.  The  lower  compartment  is  filled  with  ether 
and  mercury.  The  lower  end  of  the  inlet  tube  (A)  has  a  wedge-shaped  slit. 
The  gas  from  the  supply  pipe  passing  through  the  tube  (A)  is  conducted 
through  the  slit-like  opening  in  its  lower  end  into  the  inner  chamber  and 
passes  out  to  the  burner  through  the  elbow  (B).  When  the  temperature  is 
raised,  the  ether  and  mercury  in  the  lower  chamber  expand  and  the  mercury 
rises  in  the  upper  chamber,  gradually  restricting  the  opening  through  the 
V-shaped  slit  in  the  inlet  tube.  Thus  the  gas  supplied  to  the  burner  is 
diminished,  the  flame  reduced,  and  the  temperature  again  falls.  The  tem- 
perature can  be  arbitrarily  adjusted  by  raising  or  lowering  the  inlet  tube. 
A  scale  at  the  upper  end  of  the  inlet  tube  allows  exact  adjustment.  Complete 


192 


BIOLOGY  AND  TECHNIQUE 


shutting  off  of  the  gas  is  prevented  by  a  small  circular  opening  placed  in 
the  inlet  tube  just  above  the  slit. 

Another  cheaper  and  simpler  thermo-regulator  is  shown  in  Fig.  24.  This 
consists  of  a  long  tube  open  at  the  top  and  fitted  about  1^  inches  from 
the  top  with  two  hollow  glass  elbows.  One  of  these  elbows  remains  open, 
the  other,  situated  on  a  slightly  lower  level,  is  closed  by  a  brass  screw-cap. 
The  tube  is  filled  with  mercury  to  a  point  slightly  above  the  level  of  the 
elbow  containing  the  screw-cap.  The  height  of  the  mercury  can  thus  be 
increased  or  decreased  by  screwing  in  or  out  upon  the  cap.  Into  the  upper 
end  of  the  tube  there  is  fitted  another  device  which  consists  of  a  T-shaped 
system  of  glass  tubes,  one  arm  of  the  T  being  open  and  the  other  closed, 
the  perpendicular  leg  of  the  T  tapering  to  a  minute  opening  at  the  bottom. 


FIG.  24. — MOITESSIER  GAS  PRESSURE  REGULATOR. 


The  gas  passes  into  one  arm  of  the  T  down  through  the  tapering  leg  and 
into  the  space  immediately  above  the  mercury.  It  then  passes  out  through 
the  open  elbow  of  the  main  tube.  As  the  mercury  rises,  it  gradually 
diminishes  the  space  between  its  surface  and  the  small  opening  in  the 
tapering  tube  above  it,  finally  completely  shutting  off  the  gas  from  this 
source.  Gas  can  now  pass  only  through  a  minute  hole  perforating  the 
vertical  leg  of  the  T  an  inch  above  its  end.  The  flame  decreases  and  the 
temperature  again  sinks. 

Since  gas  pressure  in  laboratories  is  apt  to  vary,  it  is  convenient 
to  interpose  between  the  gas  supply  and  thermo-regulator  some  one 
of  the  various  forms  of  gas-pressure  regulators.  The  use  of  these 
is  not  absolutely  necessary  but  aids  considerably  in  the  maintenance 
of  a  constant  temperature.  The  one  most  commonly  employed  is  the 
so-called  Moitessier  apparatus.  This  consists  of  a  cylindrical  metal 
chamber  within  which  there  is  fitted  an  inverted  metal  bell.  Glycerin 


METHODS  USED  IN  CULTIVATION  OF  BACTERIA 


193 


is  poured  into  the  cylinder  to  the  depth  of  about  two  inches.  An 
inlet  pipe  conducts  gas  into  the  open  space  between  the  top  of  the 
glycerin  and  the  bell.  From,  the  top  of  the  bell  is  suspended  a 
conical  piece  of  metal  which  hangs  free  in  the  outlet  pipe.  As  the 
gas  pressure  under  the  bell  increases,  this  is  raised  and  the  opening 
of  the  outlet  pipe  is  gradually  diminished  by  the  cone.  Thus  the 
relation  between  the  pressure  in  the  inlet  pipe  and  the  actual  quan- 
tity of  gas  passing  through  is  equalized.  A  cup  connected  to  the 
top  of  the  bell  through  the  roof  of  the  cylinder  by  a  bar  can  be 
filled  with  birdshot  and  the  pressure  against  the  gas  can  thus  be 
modified  to  conform  with  existing  conditions. 

Colony  Study. — Cultures  are  usually  incubated  for  from  twelve 


FIG.  25. — VARIATIONS  IN  THE  CONFORMATION  OP  THE  BORDERS  OP  BACTERIAL 

COLONIES. 

to  forty-eight  hours.  Considerable  aid  to  the  recognition  of  species 
is  derived  from  the  observation  of  both  the  speed  of  growth  and 
the  appearance  of  the  colonies.  It  is  therefore  necessary  to  proceed 
in  the  study  of  developed  colonies  in  a  systematic  way.  The  develop- 
ment of  colonies  should  be  observed  in  all  cases  both  upon  gelatin 
and  upon  agar.  In  forming  any  judgment  about  colonies,  the  acidity 
or  alkalinity,  and  the  special  constitution  of  the  media  should  be 
taken  into  account.  The  colonies  are  carefully  examined  with  a 
hand  lens  and  with  the  low  power  (Leitz  No.  2,  Zeiss  A  A,  Ocular 
No.  2)  of  the  microscope.  The  colonies  should  be  observed  as  to 
size,  outline,  transparency,  texture,  color,  and  elevation  from  the 
surface  of  the  media.  Much  information,  also,  can  be  obtained  by 
observing  whether  a  colony  appears  dry,  mucoid,  or  glistening,  like 
a  drop  of  moisture.  By  a  careful  observation  of  these  points,  definite 


194  BIOLOGY  AND  TECHNIQUE 

differentiation,  of  course,  can  not  usually  be  made,  but  much  cor- 
roborative evidence  can  be  obtained  which  may  guide  us  in  the 
methods  to  be  adopted  for  further  identification  and  for  a  final 
summing  up  of  species  characteristic  as  a  whole. 

The  Counting  of  Bacteria. — It  is  often  necessary  to  determine  the 
number  of  bacteria  per  c.c.  contained  in  water,  milk,  or  other  sub- 
stances. For  this  purpose  definite  quantities  of  the  material  to  be 
analyzed  are  mixed  with  gelatin  or  agar  and  poured  into  Petri 
plates.  The  exact  dilutions  of  the  suspected  material  must  largely 
depend  upon  the  number  of  germs  which  one  expects  to  find  in  it. 
The  plates,  if  prepared  with  gelatin,  are  allowed  to  develop  at  room 
temperature  for  twenty-four  to  forty-eight  hours.  If  agar  has  been 
used,  they  are  usually  placed  in  the  incubator  at  37.5°  C.  At  the 
end  of  this  time,  the  colonies  which  have  developed  are  enumerated. 
For  this  purpose,  a  Petri  dish  is  placed  upon  a  Wolffhiigel  plate. 
This  plate  consists  of  a  disk  or  square  of  glass  which  is  divided 
into  small  squares  of  one  square  centimeter  each.  Diagonal  lines 
of  these  squares  running  at  right  angles  to  each  other  are  subdivided 
into  nine  divisions  each  in  order  to  facilitate  counting  when  the 
colonies  are  unusually  abundant.  The  Petri  dish  is  placed  upon  the 
plate  in  such  a  way  that  the  center  of  the  dish  corresponds  to  the 
center  of  the  plate.  The  colonies  in  a  definite  number  of  squares 
are  then  counted.  The  greater  the  number  of  squares  that  are 
counted  the  more  accurate  the  estimation  will  be.  When  the  growth 
is  so  abundant  that  only  a  limited  number  of  squares  can  be  counted, 
these  should  be  chosen  as  much  as  possible  from  different  parts  of 
the  plate,  and  in  practice  one  counts  usually  six  squares  in  one 
direction  and  six  at  right  angles  to  these,  so  as  to  preclude  errors 
arising  from  unequal  distribution.  The  final  calculation  is  then 
made  by  ascertaining  the  average  number  of  colonies  contained  in 
each  square  centimeter.  If  standard  Petri  dishes  have  been  used, 
this  is  multiplied  by  63.6,  the  number  of  squares  in  the  area  of  the 
dish,  and  then  by  the  dilution  originally  used. 

Thus  if  twelve  squares  have  been  counted  with  a  total  number 
of  one  hundred  and  forty-four  colonies — the  average  for  each  square 
is  twelve.  Twelve  times  63.6  is  763.2,  which  represents  the  total 
number  of  colonies  in  the  plate.  Now  if  0.1  c.c.  of  the  original 
material  (water  or  milk)  has  been  plated,  this  material  may  be 
assumed  to  have  contained  10  X  763.2,  or  7,632  bacteria  to  each 
cubic  centimeter. 


METHODS   USED  IN   CULTIVATION   OF  BACTERIA 


195 


If  dishes  of  an  unusual  size  are  employed,  the  square  area  must 
be  ascertained  by  measuring  the  radius  and  multiplying  its  square 
by  TT  (w  X  R2  =  area)  (IT  =  3.141592). 

The  number  of  bacteria  in  a  given  volume  of  a  suspension  can 
be  estimated  by  a  variety  of  methods  without  cultivation.  The  one 
most  commonly  used  is  the  method  developed  by  Wright,  which 
consists  in  mixing  a  small  measured  amount  of  the  bacterial  suspen- 
sion with  an  equal  volume  of  a  red  blood  cell  suspension  in  which 


\ 


S/ 


FlG.  26. — WOLFFHUGEL  COUNTING  PLATE. 

the  number  of  erythrocytes  per  cubic  millimeter  are  known.  Smears 
are  made  and  the  relative  number  of  bacteria  and  red  blood  cells 
per  one  or  two  hundred  fields  are  counted.  A  simple  calculation 
can  then  be  made.  This  method  has  been  described  in  the  section 
dealing  with  opsonin  technique. 

Another  useful  technique  is  the  direct  counting  of  dilutions  of 
the  bacterial  suspension,  unstained  or  stained  with  methylene  blue, 
in  a  modification  of  the  Thoma  Zeiss  counting  chamber,  known  as 
the  Helber  chamber. 


CHAPTER   IX 

METHODS  OF  DETEEMINING  BIOLOGICAL  ACTIVITIES  OF  BACTERIA 
ANIMAL  EXPERIMENTATION 

Gas  Formation. — Bacteria  of  many  varieties  produce  gas  from 
the  protein  and  the  carbohydrate  constituents  of  their  environment. 

Gas  formation  can  be  observed  in  a  very  simple  manner  by  mak- 
ing stab  cultures  in  gelatin  or  agar  containing  the  fermentable 
nutrient  substances.  In  such  cultures  bubbles  of  gas  will  form  along 
the  track  of  the  inoculation,  or,  in  the  case  of  such  semisolid  media 
as  the  tube  medium  of  Hiss,  will  spread  throughout  the  tube.  In 
the  case  of  some  anaerobes  gas  formation  in  stab  cultures  will  occur 
to  such  an  extent  that  the  medium  will  split  and  break.  It  should 
be  borne  in  mind  in  carrying  out  such  methods  that  air  is  readily 
carried  into  the  medium  with  the  inoculating  needle  or  loop  by 
splitting  of  the  medium,  also  that  media  which  have  been  stored 
in  the  cold  may  absorb  air.  Expansion  of  the  air  in  such  tubes 
may  simulate  small  amounts  of  gas  formation  and  lead  to  error. 
It  is  advisable,  therefore,  whenever  making  stab  inoculations  with 
the  above  purpose,  to  heat  the  media  and  rapidly  cool  them  before 
use. 

A  more  accurate  method  of  gas  determination  is  by  the  use  of 
fermentation  tubes,  such  as  those  devised  by  Smith.  The  gas  which 
is  formed  collects  in  the  closed  arm  of  the  fermentation  tube  and 
may  be  quantitatively  estimated.  The  fermentation,  with  gas 
production,  of  certain  substances  such  as  carbohydrates,  may  be 
determined  by  adding  these  materials  in  a  pure  state  to  the  media 
before  inoculation  with  organisms. 

In  the  case  of  carbohydrates  this  method  has  proved  of  great 
differential  value,  since  the  power  of  splitting  specific  carbohydrates 
with  gas  production  is  a  species  characteristic  of  great  constancy 
for  many  forms  of  bacteria. 

ANALYSIS  OF  GAS  FORMED  BY  BACTERIA. — Carbon  Dioxide. — Foi 
the  estimation  both  qualitatively  and  roughly  quantitatively  of  car- 

196 


DETERMINING   BIOLOGICAL   ACTIVITIES   OF   BACTERIA     197 


bon  dioxide  produced  by  bacteria,  cultures  are  grown  in  fermen- 
tation tubes  containing  sugar-free  broth,  (see  page  150)  to  which  one 
per  cent  of  pure  dextrose,  lactose,  saccharose,  or  other  sugars  has 
been  added.  The  tubes  are  incubated  until  the  column  of  gas 
formed  in  the  closed  arm  no  longer  increases  (twenty-four  to  forty- 
eight  hours).  The  level  of  the  fluid  in  the  closed  arm  is  then 
accurately  marked  and  the  column  of  gas  measured. 

The  bulb  of  the  fermentation  tube  is  then  completely  filled  with 
^  NaOH  solution,  the  mouth  closed  with  a  clean  rubber  stopper, 
and,  the  bulb  inverted  several  times  in  order  to  mix  the  gas  with 


rr 


FIG.  27. — TYPES  OF  FERMENTATION  TUBES. 

the  soda  solution.  The  tube  is  then  again  placed  in  the  upright 
position,  allowing  the  gas  remaining  to  collect  in  the  closed  arm. 
The  gas  lost  may  be  roughly  estimated  as  consisting  of  C02. 

Hydrogen. — The  gas  remaining,  after  removal  of  the  C02  in  the 
preceding  -experiment,  at  least  when  working  with  carbohydrate 
solutions,  may  be  estimated  as  hydrogen.  When  allowed  to  collect 
near  the  mouth,  further  evidence  of  its  being  hydrogen  may  be 
gained  by  exploding  it  with  a  lighted  match. 

Hydrogen  Sulphid  (H2S,  Sulphuretted  hydrogen). — In  alkaline 
media,  sulphuretted  hydrogen,  if  formed,  will  not  collect  as.  gas, 
but  will  form  a  sulphid  with  any  alkali  in  the  solution.  For  the 
estimation  of  the  formation  of  hydrogne  sulphid,  bacteria  are  cul- 


198  BIOLOGY  AND  TECHNIQUE 

tivated  in  a  strong  pepton  solution  to  which  0.1  c.c.  of  a  one  per 
cent  solution  of  ferric  tartrate  or  lead  acetate  has  been  added.  The 
addition  of  these  substances  gives  rise  to  a  yellowish  precipitate 
in  the  bottom  of  the  tubes.  If,  on  subsequent  inoculation,  the 
bacteria  produce  H2S,  this  precipitate  will  turn  black.  The  solution 
recommended  by  Pake  for  this  test  is  prepared  as  follows : 

1.  Weigh  out  30  grams  of  pepton  and  emulsify  in  200  c.c.  of  tap  water 
at  60°  C. 

2.  Wash  into  a  liter  flask  with  80  c.c.  tap  water. 

3.  Add  sodium  chlorid  5  grams  and  sodium  phosphate  3  grams. 

4.  Heat  at  100°  C.  for  30  minutes,  to  dissolve  pepton. 

5.  Filter  through  paper. 

6.  Fill  into  tubes,  10  c.c.  each,  and  to  each  tube  add  0.1  c.c.  of  a  one 
per  cent  solution  of  ferric  tartrate  or  lead  acetate.     These  solutions  should 
be  neutral. 

7.  Sterilize.1 

ACCURATE  QUANTITAVE  GAS  ANALYSES  of  bacterial  cultures  can  be 
made  only  by  the  more  complicated  methods  used  in  chemical  labora- 
tories for  quantitative  gas  analysis.  The  gas,  in  such  cases,  is 
collected  in  a  bell  jar  mounted  over  mercury,  and  subjected  to 
analysis  by  the  usual  method  described  in  works  on  analytical 
chemistry. 

Acid  and  Alkali  Formation  by  Bacteria. — Many  bacteria  produce 
acid  or  alkaline  reactions  in  culture  media,  their  activity  in  this 
respect  depending  to  a  large  extent  upon  the  nature  of  the  nutrient 
material.  Many  organisms  which  on  carbohydrate  media  produce 
acid  will  give  rise  to  alkali  if  cultivated  upon  media  containing  only 
proteins. 

Information  as  to  the  production  of  acid  or  alkali  can  be  obtained 
by  the  addition  of  one  of  a  variety  of  indicators  to  neutral  media. 
The  indicators  most  often  employed  for  this  purpose  are  litmus, 
neutral  red,  China  blue  and  the  so-called  Andrade  indicator.  An- 
drade  consists  of  100  c.c.  of  a  0.5%  aqueous  solution  of  acid  fuchsin 
to  which  16  c.c.  of  accurately  normal  NaOH  has  been  added. 
Changes  in  the  color  of  these  indicators  show  whether  acids  or 
alkalis  have  been  produced. 

Great  help  in  differentiation  is  obtained  by  adding  chemically 
pure  carbohydrates  to  media  to  which  litmus  has  been  added,  and 


1  Quoted  from  Eyre,  ' '  Bact.  Technique, ' '  Phila.,  1903. 


DETERMINING   BIOLOGICAL   ACTIVITIES   OF   BACTERIA     199 

then  determining  whether  or  not  acid  is  formed  from  these  substances 
by  the  microorganisms.  These  tests  have  been  of  special  importance 
in  the  differentiation  of  the  typhoid-colon  groups  of  bacilli. 

Quantitative  estimation  of  the  degree  of  acidity  or  alkalinity 
produced  by  bacteria  may  be  made  by  careful  titration  of  definite 
volumes  of  the  medium  before  and  after  bacterial  growth  has  taken 
place. 

The  variety  of  acid  formed  by  bacteria  depends  largely  upon  the 
nature  of  the  nutrient  medium.  The  acids  most  commonly  resulting 
from  bacterial  growth  are:  lactic,  acetic,  oxalic,  formic,  and  hippuric 
acids.  Qualitative  and  quantitative  estimation  of  these  acids  may 
be  made  by  any  of  the  methods  employed  by  analytical  chemists. 

Indol  Production  by  Bacteria. — Many  bacteria  possess  the  power 
of  producing  indol.  Though  formerly  regarded  as  a  regular  accom- 
paniment of  protein  decomposition,  later  researches  have  shown  that 
indol  production  is  not  always  coexistent  with  putrefaction  processes 
and  occurs  only  when  pepton  is  present  in  the  pabulum. 

Indol  formation  by  bacteria  is  determined  by  the  so-called  nitroso- 
indol  reaction.  Organisms  are  grown  in  sugar-free  pepton  broth 
or  in  the  pepton-salt  bouillon  of  Dunham.  (See  page  151.)  Media 
containing  fermentable  substances  are  not  favorable  for  indol 
production  since  acids  interfere  with  its  formation.  The  cultures 
are  usually  incubated  for  three  or  four  days  at  37°  C.  At  the  end 
of  this  time,  ten  drops  of  concentrated  sulphuric  acid  are  run  into 
each  tube.  If  a  pink  color  appears,  indol  is  present,  and  we  gather 
the  additional  information  that  the  microorganism  in  question  has 
been  able  to  form  nitrites  by  reduction  (e.g.,  cholera  spirillum).  If 
the  pink  color  does  not  appear  after  the  addition  of  the  sulphuric 
acid  alone,  nitrites  must  be  supplied.  This  is  done  by  adding  to 
the  fluid  about  1  c.c.  of  a  0.01  per  cent  aqueous  solution  of  sodium 
nitrite.  The  sodium  nitrite  solution  does  not  keep  for  any  length 
of  time  and  should  be  freshly  made  up  at  short  intervals. 

VANILLIN  TEST.2 — An  excellent  test  for  indol  is  the  so-called 
vanillin  test.  To  5  c.c.  of  the  culture  add  5  drops  of  5  per  cent 
vanillin  solution  in  95  per  cent  alcohol,  and  2  c.c.  of  concentrated 
hydrochloric  acid  or  sulphuric  acid.  If  indol  is  present,  an  orange 
color  develops  within  2  or  3  minutes.  Tryptophane  gives  a  reddish 


-  Stcensma,  Zeit.  £.  Physiol,  Chem.,  47,   1906;    Nelson,  Jour,   of  Biol.   Chem., 
24,  1916. 


200  BIOLOGY  AND  TECHNIQUE 

violet  which  grows  deeper  when  the  medium,  is  heated  or  allowed 
to  stand. 

Phenol  Production  by  Bacteria. — Phenol  is  often  a  by-product  in 
the  coarse  of  protein  cleavage  by  bacteria.  To  determine  its  presence 
in  cultures,  bacteria  are  cultivated  in  flasks  containing  about  50-100 
c.c.  of  nutrient  broth.  After  three  to  four  days'  growth  at  37°  C., 
5  c.c.  of  concentrated  HC1  are  added  to  the  culture,  the  flask  is 
connected  with  a  condenser,  and  about  10-20  c.c.  are  distilled  over. 

To  the  distillate  may  be  added  0.5  c.c.  of  Milloii's  reagent  (solu- 
tion of  mercurous  nitrate  in  nitric  acid),  when  a  red  color  will 
indicate  phenol;  or  0.5  c.c.  of  a  ferric  chloride  solution,  which  will 
give  a  violet  color  if  phenol  is  present. 

Reducing  Powers  of  Bacteria. — The  power  of  reduction,  possessed 
by  many  bacteria,  is  shown  by  their  ability  to  form  nitrites  from 
nitrates.  This  is  easily  demonstrated  by  growing  bacteria  upon 
nitrate  broth  (see  page  151).  Bacteria  are  transferred  to  test  tubes 
containing  this  solution  and  allowed  to  grow  in  the  incubator  for 
four  or  five  days.  The  presence  of  nitrites  is  then  chemically 
determined.3 

In  bacteriological  work,  4  c.c.  of  the  culture  fluid  are  poured  into 
a  clean  test  tube,  and  to  it  are  gradually  added  2  c.c.  of  the  mixed 
test  solutions.  A  pink  color  indicates  the  presence  of  nitrites,  the 
intensity  of  the  color  being  proportionate  to  the  amount  of  nitrite 
present. 

The  reducing  powers  of  bacteria  may  also  be  shown  by  their 
ability  to  decolorize  litmus,  methylene-blue,  and  some  other  anilin 
dyes,  which  on  abstraction  of  oxygen  form  colorless  leukobases. 

8  We  are  indebted  to  Dr.  J.  P.  Mitchell,  of  Stanford  University,  for  the  follow- 
ing technique  for  nitrite  tests: 

I.  Sulphanilic  Acid. — Dissolve  0.5  g.  in  150  c.c.  of  acetic  acid  of  Sp.  Gr.  1.04. 
(Acetic  acid  of  1.04  prepared  by  diluting  400  c.c.  of  cone,  of  sp.  gr.  1.75  with 
700.  c.c.  of  water.) 

II.  A-Naphthylamin. — Dissolve  0.1  gr.  in  20  c.c.  of  water,  boil,  filter  (if  neces- 
sary), and  to  clear  filtrate  add  180  c.c.  of  acetic  acid,  Sp.  Gr.  1.04. 

The  solutions  are  kept  separate  and  mixed  in  equal  parts  just  before  use. 

In  carrying  out  the  test,  put  2  c.c.  of  each  reagent  in  a  test  tube  and  add 
substance  to  be  tested.  (In  ordinary  water  analysis  use  100  c.c.)  Cover  tube 
with  watch  glass  and  sot  in  warm  water  for  20  minutes.  Observe  presence  or 
absence  of  pink  color  promptly.  Always  run  a  blank  on  the  distilled  water  used 
for  rinsing  to  avoid  errors  due  to  nitrites  in  the  water,  or  in  the  air  of  the 
laboratory. 


DETERMINING  BIOLOGICAL  ACTIVITIES  OF  BACTERIA      201 

Enzyme  Action.4 — The  action  of  the  enzymes  produced  by  bac- 
teria may  be  demonstrated  by  bringing  the  bacteria,  or  their  isolated 
ferments,  into  contact  with  the  proper  substances  and  observing 
both  the  physical  and  chemical  changes  produced.  In  obtaining 
enzymes  free  from  living  bacteria,  it  is  convenient  to  kill  the  cultures 
by  the  addition  either  of  toluol  or  of  chloroform.  Both  of  these 
substances  will  destroy  the  bacteria  without  injuring  the  enzymes. 
Enzymes  may  also  be  obtained  separate  from  the  bodies  of  the 
bacteria  by  nitration. 

PROTEOLYTIC  ENZYMES. — The  most  common  evidences  of  proteo- 


T 


FIG.  28. — TYPES  OF  GELATIN  LIQUEFACTION  BY  BACTERIA. 

lytic  enzyme  action  observed  in  bacteriology  are  the  liquefaction 
of  gelatin,  fibrin  or  coagulated  blood-serum,  and  the  peptonization 
of  milk.  This  may  be  observed  both  by  allowing  the  proper  bacteria 
to  grow  upon  these  media,  or  by  mixing  sterilized  cultures  with 
small  quantities  of  these  substances.5  The  products  of  such  a  reac- 
tion may  be  separated  from  the  bacteria  by  filtration  and  then  tested 
for  pepton  by  the  biuret  reaction. 


4  See  also  pp.  54  et  seq. 

8  Bitter,  Archiv  f.  Hyg.,  v.  1886. 


202  BIOLOGY  AND  TECHNIQUE 

Proteolytic6  enzymes  may  also  be  determined  by  growing  the 
bacteria  upon  fluid  media  containing  albumin  solutions,  blood  serum, 
or  milk  serum,  then  precipitating  the  proteins  by  the  addition  of 
ammonium  sulphate  (about  30  grams  to  20  c.c.  of  the  culture  fluid) 
and  warming  between  50  to  60°  C.  for  thirty  minutes.  The  pre- 
cipitate is  then  filtered  off,  the  filtrate  made  strongly  alkalm  with 
NaOH,  and  a  few  drops  of  copper  sulphate  solution  added.  A  violet 
color  indicates  the  presence  of  pepton — proving  proteolysis  of  the 
original  albumin. 

DIASTATIC  ENZYMES. — The  presence  of  diastatic  ferments  may  be 
determined  by  mixing  broth  cultures  of  the  bacteria  with  thin  starch 
paste.  It  is  necessary  that  both  the  cultures  and  the  starch  paste 
be  absolutely  free  from  sugar.  After  remaining  in  the  incubator  for 
five  or  six  hours,  the  fluid  is  filtered  and  the  filtrate  tested  by  methods 
used  for  determining  the  presence  of  sugars. 

INVERTING  FERMENTS. — Inverting  ferments  are  determined  by  a 
procedure  similar  to  the  above  in  principle.  Dilute  solutions  of  cane 
sugar  are  mixed  with  old  cultures  or  culture  filtrates  of  the  respec- 
tive bacteria  and  the  mixture  allowed  to  stand.  It  is  then  filtered, 
and  the  filtrate  tested  for  glucose,  preferably  by  Fehling's  solution. 

ANIMAL   EXPERIMENTATION 

In  the  study  of  pathogenic  microorganisms,  animal  experimenta- 
tion is  essential  in  many  instances.  The  virulence  of  any  given 
organism  for  a  definite  animal  species  and  the  nature  of  the  lesions 
produced  are  characteristics  often  of  great  value  in  differentiation. 
Isolation,  moreover,  of  many  bacteria  is  greatly  facilitated  by  the 
inoculation  of  susceptible  animals  and  recovery  of  the  pathogenic 
organism  from  the  heart's  blood  or  from  the  lesions  produced  in 
various  organs.  That  investigations  into  the  phenomena  of  im- 
munity would  be  absolutely  impossible  without  the  use  of  animal 
inoculation  is,  of  course,  self-evident,  for  by  this  method  only  can 
the  action  of  bacteria  in  relation  to  living  tissues,  cells,  and  body- 
fluids  be  observed. 

The  animals  most  commonly  employed  for  such  observations  are 
guinea-pigs,  white  mice,  white  rats,  and  rabbits.  The  method  of 
inoculation  may  be  either  subcutaneous,  intrapleural,  intraperitoneal, 

*Hanlcin  and  WesfbrooTc,  Ann.  Past.,  vi.,  1892. 


DETERMINING   BIOLOGICAL   ACTIVITIES   OF   BACTERIA     203 

intravenous,  or  subdural,  etc.  It  must  be  borne  in  mind  always 
that  the  mode  of  inoculation  may  influence  the  course  of  an  infection 
no  less  than  does  the  virulence  of  the  microorganism  or  the  size 
of  the  dose. 

Inoculations  are  made  with  some  form  of  hypodermic  needle 
fitted  to  a  syringe.  The  most  convenient  syringes  are  the  all-glass 
Luer  or  the  Debove  syringes,  which,  however,  are  expensive.  Any 
form  of  sterilizable  syringe  may  be  used.  In  making  inoculations 
the  hair  of  the  animal  should  be  clipped  and  the  skin  disinfected 
with  carbolic  acid  or  alcohol. 

Subcutaneous  inoculations  are  most  conveniently  made  in  the 
abdominal  wall,  where  the  skin  is  thin.  After  clipping  and  steriliz- 
ing, the  skin  is  raised  between  the  fingers  of  the  left  hand  and  the 
needle  plunged  in  obliquely  so  as  to  avoid  penetrating  the  abdominal 
wall  and  entering  the  peritoneum. 

In  making  intraperitoneal  inoculations,  great  care  must  be  exer- 
cised not  to  puncture  the  gut.  This  can  be  avoided  by  passing  the 
needle  first  through  the  skin  in  an  oblique  direction,  then  turning 
it  into  a  position  more  vertical  to  the  abdomen  and  perforating  the 
muscles  and  peritoneum  by  a  very  short  and  carefully  executed  stab. 

Intravenous  inoculations  in  rabbits  are  made  into  the  veins  run- 
ning along  the  outer  margins  of  the  ears.  The  hair  over  the  ear 
is  clipped  and  the  animal  held  for  a  short  time  head  downward 
so  that  the  vessels  of  the  head  may  fill  with  blood.  An  assistant 
holds  the  animal  firmly  in  a  horizontal  position,  the  operator  grasps 
the  tip  of  the  ears  with  the  left  hand,  and  carefully  passes  his  needle 
into  the  vein  in  the  direction  as  nearly  as  possible  parallel  to  its 
course. 

Mice  are  usually  inoculated  under  the  skin  near  the  base  of 
the  tail.  They  may  be  placed  in  a  jar  over  which  a  cover  of  stiff 
wire-gauze  is  held.  They  are  then  grasped  by  the  tail,  by  which 
they  are  drawn  up  between  the  side  of  the  jar  and  the  edge  of  the 
wire  cover,  so  that  the  lower  end  of  the  back  shall  be  easily  acces- 
sible. The  skin  is  then  wiped  with  a  piece  of  cotton  dipped  in 
carbolic  solution  and  the  needle  is  inserted.  Great  care  must  be 
exercised  to  avoid  passing  the  needle  too  close  to  the  vertebral 
column.  Mice  are  extremely  delicate,  and  any  injury  to  the  spine 
usually  causes  immediate  death. 

With  proper  care  mice  or  rats  may  be  easily  injected  intra- 
venously if  a  sufficiently  fine  needle  is  used.  There  are  four  super- 


204  BIOLOGY  AND  TECHNIQUE 

ficially  placed  veins  running  along  the  tail,  which  stand  out 
prominently  when  rubbed  with  cotton  moistened  with  xylol.  Into 
these  the  injections  are  made. 

When  inoculating  rats  or  guinea-pigs  with  Bacillus  pestis  the 
Kolle  vacination  method  is  used.  The  skin  is  merely  shaved  and  a 
loopful  of  the  culture  vigorously  rubbed  into  the  shaven  area. 

The  various  forms  of  animal  holders  which  have  been  devised 
are  rarely  necessary  in  bacteriological  work  unless  working  un- 
assisted, immobilization  of  the  animals  being  easily  accomplished 
by  the  hands  of  a  skilled  assistant. 

Autopsies  upon  infected  animals  must  be  carefully  made.  The 
animals  are  tied,  back  down,  upon  pans  fitted  in  the  corners  with 
clamps  for  the  strings.  They  are  then  moistened  either  with  hot 
water  or  with  a  weak  solution  of  carbolic  acid,  so  that  contamination 
by  hair  may  be  avoided.  A  median  cut  is  made,  the  skin  is  carefully 
dissected  back,  and  the  body  cavities  are  opened  with  sterile  instru- 
ments. Cultures  may  then  be  taken  from  exudates,  blood,  or  organs 
under  precautions  similar  to  those  recommended  below  for  similar 
procedures  at  autopsy  upon  man. 

Inoculated  animals  should  be,  if  possible,  kept  separate  from 
healthy  animals.  Rabbits  and  guinea-pigs  are  best  kept  in  gal- 
vanized iron-wire  cages,  which  are  fitted  with  floor-pans  that  can 
be  taken  out  and  cleaned  and  sterilized.  Mice  may  be  kept  in 
battery  jars  fitted  with  perforated  metal  covers.  The  mice  should 
be  supplied  with  large  pieces  of  cotton  upon  batting  since  they  are 
delicately  susceptible  to  cold. 

The  Bleeding  of  Animals. — Animals  are  bled  for  the  purpose  of 
obtaining  either  corpuscles,  defibrinated  blood  or  serum. 

In  order  to  obtain  small  amounts  of  blood,  that  is  about  5  or 
10  c.c.,  from  rabbits,  the  ear  is  shaved  and  had  best  be  immersed 
in  warm  water  for  a  few  moments  in  order  to  expand  the  vessels. 
Gentle  rubbing  with  alcohol  is  also  advantageous.  The  rabbit  is 
then  held  with  head  hanging  downward,  and  a  broad  needle  of  the 
Hagedore  needle  type  is  thrust  into  the  vein  and  withdrawn.  The 
drops  can  be  caught  directly  in  a  centrifuge  tube,  or  in  the  culture 
media  for  which  it  is  intended.  All  blood  media  should  be  incubated 
for  24  hours  and  the  contaminated  tubes  discarded. 

A  better  method  is  to  take  blood  from  rabbits  and  from  guinea 
pigs  directly  from  the  heart.  If  this  is  skillfully  done  the  animals 
can  be  repeatedly  bled  without  being  killed.  For  taking  blood  for 


DETERMINING   BIOLOGICAL   ACTIVITIES   OF    BACTERIA     205 

complement  in  Wassermann  reactions,  this  is  among  the  best 
methods  since  large  guinea  pigs  can  be  alternately  bled  and  rested. 
Both  in  rabbits  and  guinea  pigs,  bleeding  directly  from  the  heart 
is  easily  accomplished  after  a  little  practice.  The  anterior  thorax 
of  the  animal  is  clipped  and  painted  with  tincture  of  iodin  and  the 
operator  in  feeling  for  the  third  interspace  close  to  the  sternum 
had  best  paint  the  tips  of  his  fingers  with  iodin.  A  twenty-two 
gauge  needle  about  two  inches  long  is  then  attached  to  a  syringe 
and  passed  downward  in  the  third  left  interspace  close  to  the 
sternum,  slight  suction  being  exercised  at  the  same  time.  There 
is  not  much  purpose  in  describing  this  in  detail  since  it  can  be 
taught  only  by  practice. 

Both  rabbits  and  guinea  pigs  can  be  bled  from  the  carotid.  The 
animal  is  anesthetized  as  above,  and  the  carotid  laid  bare.  It  is 
found  vety  close  to  the  trachea,  in  rabbits  lying  almost  in  contact 
with  the  trachea,  and  a  little  behind  it.  It  is  carefully  separated 
from  the  vagus  nerve,  and  tied  off  in  its  distal  portion.  The  thread 
with  which  it  is  tied  can  be  used  to  handle  it  thereafter.  A  sterile 
glass  cannula  can  be  thrust  into  the  artery  and  the  blood  taken 
through  this,  or  else,  as  we  prefer  to  do  it,  the  side  of  the  artery 
is  picked  up  with  a  very  fine  forceps  and  held  with  one  hand  while 
it  is  cut  across  with  a  sharp  scissors.  In  this  way  the  blood  can 
be  directed  straight  into  a  wide  mouth  flask  without  being  allowed 
to  come  in  contact  with  anything  until  it  hits  the  inside  of  the  flask. 

Larger  animals,  like  sheep,  goats,  horses,  are.  easily  bled  by 
plunging  a  sterile  needle  into  the  external  jugular  vein  which  runs 
in  these  animals  from  a  line  just  behind  the  angle  of  the  lower  jaw 
to  the  sterno-clavicular  junction. 

The  blood  can  be  run  directly  into  media  as  for  blood  agar,  blood 
broth  and  chocolate  medium. 

If  serum  is  desired,  it  can  be  run  into  containers  of  various  kinds 
slanted  and  allowed  to  clot  in  the  ice-box. 

If  defibrinated  blood  is  desired,  the  blood  can  be  taken  directly 
into  sterile  flasks  containing  pieces  of  broken  glass  or  beads  and 
attenuated  before  clot.  Such  blood  can  be  kept  in  the  ice-chest  and 
added  to  media  subsequently. 

Blood  can  be  also  preserved  for  culture  purposes  by  the  addition 
of  just  enough  ether  to  hemolyze  it,  and  added  to  media  in  this 
form.  The  ether  is  evaporated  off. 


CHAPTER   X 

THE    BACTEEIOLOGICAL    EXAMINATION    OF    MATEEIAL    FEOM    PA- 
TIENTS  AND    AN    OUTLINE    OF    THE    BACTERIAL    FLORA    OF 
THE  NORMAL  HUMAN  BODY 

TECHNICAL  procedures  for  the  examination  of  specimens  of 
exudates,  stools,  sputum,  etc.,  in  various  conditions  are  given  in 
appropriate  places  in  the  text  dealing  with  the  individual  diseases. 
In  this  chapter  we  wish  to  discuss  briefly  general  principles  of 
bacteriological  examination  which  will  be  useful  in  properly  col- 
lecting and  handling  materials  which  are  sent  to  the  laboratory 
for  diagnosis  or  which  the  bacteriologist  takes  from  the  patient 
himself. 

In  making  bacteriological  examinations  of  material  taken  from 
living  patients,  or  at  autopsy,  the  validity  of  result  is  as  fully 
dependent  upon  the  technique  by  which  the  material  is  collected, 
as  upon  proper  manipulation  in  the  later  stages  of  examination. 

Material  taken  at  autopsy  should  be,  if  possible,  directly  trans- 
ferred from  the  cadaver  to  the  proper  culture  media.  If  cultures 
are  to  be  taken  from  the  liver,  spleen,  or  other  organs,  the  surface 
of  the  organ  should  first  be  seared  with  a  hot  scalpel  and  an  incision 
made  through  the  capsule  of  the  organ  in  the  seared  area,  with  the 
same  instrument.  The  platinum  needle  can  then  be  plunged  through 
this  incision  and  material  for  cultivation  be  taken  with  little  chance 
of  surface  contamination.  When  blood  is  to  be  transferred  from 
the  heart,  the  heart  muscle  may  be  incised  with  a  hot  knife,  or  else 
the  needle  of  a  hypodermic  syringe  may  be  plunged  through  the 
previously  seared  heart  muscle  and  the  blood  aspirated.  The  same 
end  can  be  accomplished  by  means  of  a  pointed,  freshly  prepared 
Pasteur  pipette.  In  taking  specimens  of  blood  at  autopsy  it  is  safer 
to  take  them  from  the  arm  or  leg,  by  allowing  the  blood  to  flow 
into  a  broad,  deep  cut  made  through  the  sterilized  skin,  than  from 
the  heart,  since  it  has  been  found  that  post-mortem  contamination 
of  the  heart 's  blood  takes  place  rapidly,  probably  through  the  large 
veins  from  the  lungs.  Exudates  from  the  pleural  cavities,  the  peri- 

206 


BACTERIOLOGICAL   EXAMINATION   OF   MATERIAL  207 

cardium,  or  the  peritoneum  may  be  taken  with  a  sterilized  syringe 
or  pipette.  Under  all  circumstances  it  should  be  remembered  that 
cultures  taken  from  blood  or  tissues  of  the  cadaver  will  be  con- 
taminated, unless  cultures  are  taken  within  a  few  hours  after  death. 
Bacteria  get  into  the  circulation  and  multiply  throughout  the  body 
with  astonishing  speed  after  death. 

Materials  collected  at  the  bedside  or  in  the  operating-room  should 
be  transferred  directly  to  the  proper  media  or  else  into  sterile  test 
tubes  and  so  sent  to  the  laboratory.  When  the  material  is  scanty, 
it  may  be  collected  upon  a  sterile  cotton  swab,  which  should  be 
immediately  replaced  in  the  sterilized  containing  tube  and  sent  to 
the  laboratory. 

Syringes,  when  used  for  the  collection  of  exudates  or  blood, 
should  be  of  some  variety  which  is  easily  sterilizable  by  dry  heat, 
or  boiling.  Most  convenient  of  the  forms  in  common  use  are  the 
all-glass  "Luer"  syringe,  or  the  cheaper  "Sub-Q"  model.  Instru- 
ments which  can  be  sterilized  only  by  chemical  disinfectants  should 
not  be  used.  When  fluids  are  collected  for  bacteriological  examina- 
tion, such  as  spinal  fluid,  ascitic  fluid,  or  pleural  exudates,  it  is 
convenient  to  have  them  taken  directly  into  sterilized  centrifuge 
tubes,  since  it  is  often  necessary  to  concentrate  cellular  elements  by 
centrifugalization.  By  immediate  collection  in  these  tubes,  the 
danger  of  contamination  is  avoided. 

Examination  of  Exudates. — Pus. — Pus  should  first  be  examined 
morphologically  by  some  simple  stain,  such  as  gentian-violet,  and 
by  the  Gram  stain.  It  is  convenient,  also,  to  stain  a  specimen  by 
Jenner's  stain,  in  order  to  show  clearly  the  relation  of  bacteria  to 
the  cells.  Such  morphological  examination  not  only  furnishes  a 
guide  to  future  manipulation,  but  supplies  a  control  for  the  results 
obtained  by  cultural  methods.  Specimens  of  the  pus  are  then  trans- 
ferred to  the  proper  media,  and  pour-plates  made  or  streaks  made 
upon  the  surface  of  previously  prepared  agar  or  serum-agar  plates. 

A  guide  to  the  choice  of  media  is  often  found  in  the  result  of 
the  morphological  examination.  In  most  cases,  it  is  well  also  to 
make  anaerobic  cultures  by  some  simple  method.  (See  page  179 
et  seq.) 

The  colonies  which  develop  upon  the  plates  should  be  studied 
under  the  microscope,  and  specimens  from  the  colonies  transferred 
to  covor-glasses  and  slides  for  morphological  examination  and  to  the 
various  media  for  further  growth  and  identification.  Animal  inocu- 
lation and  agglutination  tests  must  often  also  be  resorted  to.  A 


208  BIOLOGY  AND  TECHNIQUE 

knowledge  of  the  source  of  the  material  may  furnish  considerable 
aid  in  making  a  bacteriological  diagnosis,  though  great  caution  in 
depending  upon  such  aid  is  recommended. 

If  the  morphological  examination  shows  Gram-positive  micro- 
cocci,  as  in  staphylococcus  boils,  any  ordinary  properly  made  agar 
will  suffice. 

If  streptococci  are  present  in  the  Gram  stain,  it  will  be  useful 
to  employ  blood  agar  plates  without  glucose. 

When  the  pus  is  gonorrheal,  ascitic  agar  plates  with  glucose 
should  be  used,  and  the  pus  transferred  directly  from  the  patient 
to  the  plate  and  incubated  before  it  chills.  In  the  case  of  pus  from 
abrasions  of  the  skin,  furuncles  or  boils  that  arouse  any  suspicion 
of  anthrax  a  careful  search  for  Gram-positive  bacilli  should  be  made 
with  the  Gram  stain,  and  the  characteristic  colonies  looked  for  on 
ordinary  agar  plates. 

When  plentiful  leucocytes  are  present  and  the  pus  shows  no 
organisms  in  smear,  this  should  not  discourage  culture  since  it  is 
not  unusual  to  obtain  colonies  on  culture  when  nothing  can  be 
found  by  smear. 

In  the  examination  of  peritoneal,  pericardial,  or  pleural  exudates 
it  is  often  advantageous  to  use  the  sediment  obtained  by  centrifugaliza- 
tion.  A  differential  count  of  the  cells  present  may  be  of  aid  in  confirm- 
ing the  bacteriological  findings.  Morphological  examination  and  cul- 
tural examination  are  made  as  in  the  case  of  pus.  Specimens  should 
also  in  these  cases  be  stained  for  tubercle  bacilli.  Whenever  mor- 
phological examinations  of  such  fluids  are  negative,  no  bacteria 
being  found,  and  especially  when  among  the  cellular  elements  the 
lymphocytes  preponderate,  the  search  for  tubercle  bacilli  should  be 
continued  by  means  of  animal  inoculation.  Guinea-pigs  should  be 
inoculated  intraperitoneally  with  specimens  of  the  fluid.  The 
animals  will  usually  die  within  six  to  eight  weeks,  but  can  be  killed 
and  examined  at  the  end  of  about  six  weeks  if  they  remain  alive. 
The  chances  for  a  positive  result  are  considerably  increased  if  the 
fluid  is  set  away  in  the  ice-chest  until  a  clot  has  formed  and  the 
animals  are  inoculated  with  the  material  from  the  broken-up  clot. 

Spinal  Fluid. — Normal  spinal  fluid  is  a  clear,  colorless  fluid  which 
contains  not  more  than  ten  to  twelve  cells  per  cubic  millimeter. 
Anything  above  this  should  be  regarded  as  suspicious.  When  clear 
spinal  fluid  is  brought  to  the  laboratory  it  is  always  well  to  shake 
up  the  specimen  and  do  a  direct  count. 


BACTERIOLOGICAL  EXAMINATION   OF   MATERIAL  200 

It  is  of  value  also  to  do  a  globulin  reaction  on  such  clear  fluids, 
which  is  easily  done  by  Noguchi's  butyric  acid  method  as  follows: 

To  0.2  c.c.  of  spinal  fluid  add  0.5  c.c.  of  a  10  per  cent  butyric  acid 
solution  in  physiological  salt  solution.  Boil  the  mixture  and  add  0.1  c.c. 
of  normal  sodium  hydrate,  and  boil  again.  A  flocculent  precipitate  forms 
in  positive  reactions. 

Clear  specimens  of  fluid  of  this  kind  should  be  examined  with 
an  intelligent  understanding  of  the  nature  of  the  case.  Syphilitic 
spinal  fluids  are  almost  always  clear,  but  the  cells  are  increased  to 
100  or  more  per  cubic  millimeter.  The  cells  consist  mainly  of 
lymphocytes.  Low  counts  may  be  encountered  in  tabes  and  general 
paresis.  The  determination  of  these  facts  will  be  valuable  in  con- 
nection with  the  subsequent  Wassermann  reaction  or  colloidal  gold 
reaction  on  these  fluids,  and  with  bacteriological  examination. 

In  infantile  paralysis  or  acute  poliomyelitis,  the  spinal  fluid  is 
usually  clear.  The  cells  here  are  increased  from  the  beginning. 
According  to  Peabody,  Draper  and  Dochez1  during  the  early  days 
of  the  disease,  80  or  more  per  cent  of  the  cells  may  be  polynuclear. 
After  72  hours,  however,  the  mononuclears  preponderate.  The  cell 
count  may  go  up  even  in  the  prodromal  period.  The  highest  cell 
count  is  usually  found  in  the  first  week,  gradually  coming  down 
until  the  fourth  week  of  the  disease.  The  globulin  reaction  is 
usually  highest  in  the  second  and  third  week,  but  the  writers  men- 
tioned above  found  a  percentage  of  cases  in  which  the  cell  counts 
were  normal.  These  facts  are  given  because  they  should  be  taken 
into  consideration,  together  with  bacteriological  examinations. 

Tuberculous  fluids  are  entirely  clear,  or  but  slightly  turbid. 
When  the  suspicion  of  tuberculosis  exists,  the  fluid  should  be  handled 
as  sterilely  as  possible,  and  allowed  to  stand  in  the  ice-chest  until 
a  little,  white,  thread-like  clot  appears  in  the  center,  which  sinks 
to  the  bottom  of  the  tube.  It  is  in  this  clot  that  tubercle  bacilli 
can  be  found  by  careful  search.  It  is  smeared  on  the  slide  and 
stained  by  the  usual  methods.  If  enough  fluid  is  available,  the 
residue  should  be  injected  into  one  or  two  guinea  pigs  in  as  large 
quantities  as  can  be  obtained.  The  cells  in  tuberculous  fluid  are 
chiefly  lymphocytes. 

Acute  meningitis  is  most  commonly  caused  by  the  meningococcus, 
pneumococcus,  streptococcus,  less  commonly  by  influenza  bacilli  and 

1  Peabody,  Draper  and  Dochez. 


210  BIOLOGY  AND  TECHNIQUE 

other  organisms.  Such  fluid  may  range  from  slight  turbidity  to 
thick  purulence.  The  cells  in  such  fluid  consist  almost  entirely  of 
polynuclear  leucocytes  during  the  acute  stages.  Smears  should  he 
made  immediately  and  stained  by  Gram.  If  Gram-positive  organisms 
are  present,  they  can  immediately  be  recognized,  and  cultures  taken 
accordingly.  In  epidemic  meningitis  the  Gram-negative  meningo- 
cocci  will  be  found  mostly  intracellular,  but  some  also  extracellular. 
Sometimes  a  very  prolonged  search  must  be  made  before  any 
meningococci  can  be  found,  because  these  organisms  readily  undergo 
autolysis.  In  the  fluid  from  an  acute  case  of  meningitis  in  which 
many  polynuclear  leucocytes  are  present,  and  no  organisms  can  be 
found,  it  is  pretty  safe  to  suspect  epidemic  meningitis,  since  we 
have  on  a  number  of  occasions  encountered  fluids  of  this  kind  in 
which  no  organisms  could  be  seen.  Cultures  should,  in  all  cases, 
be  taken  even  when  the  organisms  are  found,  since  this  may  be  of 
value  in  determining  the  meningococcus  type,  in  finding  out  whether 
the  particular  meningococcus  is  agglutinable  in  the  polyvalent  serum 
used,  and  the  collection  of  the  type  of  meningococci  which  are 
present  in  an  epidemic  is  a  part  of  the  bacteriologist's  contribution 
to  the  successful  production  of  sera.  The  cultures  are  best  taken 
on  plates  of  hormone  agar  with  0.5  per  cent  glucose,  and  hemolyzed 
blood  or  ascitic  fluid  added. 

Pneumococci,  streptococci,  influenza  bacilli,  etc.,  may  be  cul- 
tivated by  appropriate  methods. 

The  cytological  character  of  the  fluid  and  the  relationship  of 
cells  to  bacteria  should  always  be  determined  since  this  may  have 
a  certain  amount  of  prognostic  significance. 

Examination  of  Urine. — Bacteriological  examination  of  the  urine 
is  of  value  only  when  specimens  have  been  taken  with  sterile 
catheters,  and  care  has  been  exercised  in  the  disinfection  of  the 
external  genitals.  This  is  particularly  important  in  the  female. 
Many  of  the  numerous  finds  of  bacillus  coli  in  urine  are  unquestion- 
ably due  to  defective  methods  of  collecting  material.  Urine  should 
be  centrifugalized  and  the  sediment  examined  morphologically  and 
pour-plates  and  surface  smears  made  upon  the  proper  media. 
If  necessary,  animal  inoculation  may  be  done.  In  examining  urine 
for  tubercle  bacilli,  special  care  should  be  taken  in  staining  methods 
so  as  to  differentiate  from  Bacillus  smegmatis.  When  the  question 
is  one  of  infection  of  one  kidney  alone  the  specimens  must  of  course 
be  obtained  by  ureteral  catheterization. 


BACTERIOLOGICAL  EXAMINATION    OF   MATERIAL  211 

Examination  of  Feoes. — Human  feces  contain  an  enormous  num- 
ber of  bacteria  of  many  varieties.  Klein,2  by  special  methods, 
estimated  that  there  were  about  75,000,000  bacteria  in  one  milligram 
of  feces.  It  has  been  a  noticeable  result  of  all  the  investigations 
upon  the  feces,  that  although  enormous  numbers  can  be  counted 
in  morphological  specimens,  only  a  disproportionately  smaller  num- 
ber can  be  cultivated  from  the  same  specimen.  This  is  explicable 
upon  the  ground  that  special  culture  media  are  necessary  for  many 
of  the  species  found  in  intestinal  contents  and  upon  the  consideration 
that  many  of  the  bacteria  which  are  present  in  the  morphological 
specimen  are  dead,  showing  that  there  are  bactericidal  processes 
going  on  in  some  parts  of  the  intestinal  tract,  possibly  through  the 
agency  of  intestinal  secretions,  bile,  and  the  action  of  the  products 
of  metabolism  of  the  hardier  species  present.  By  far  the  greater 
part  of  the  intestinal  flora  consists  of  members  of  the  colon  group, 
bacilli  of  the  lactis  aerogenes  group,  Bacillus  faecalis  alkaligenes, 
Bacillus  mesentericus,  and  relatively  smaller  numbers  of  strepto- 
cocci, staphylococci,  and  Gram-positive  anaerobes.  Many  other 
species,  however,  may  be  present  without  being  necessarily  con- 
sidered of  pathological  significance.  Certain  writers  have  recently 
laid  much  stress  upon  a  preponderance  of  Gram-positive  bacteria  in 
specimens  of  feces,  claiming  that  such  preponderance  signifies  some 
form  of  intestinal  disturbance.  Herter3  has  recently  advanced  the 
opinion  that  the  presence  of  Bacillus  aerogenes  capsulatus  in  the 
intestinal  canal  is  definitely  associated  with  pernicious  anemia.  This 
is  discussed  in  another  section.  The  determination  of  these  bacilli 
in  the  stools  is  made  both  by  morphological  examination  by  means 
of  Gram  stain  and  by  isolation  of  the  bacteria.  Such  isolation  is 
easily  done  by  the  method  of  Welch  and  Nuttal.4  A  suspension  of 
small  quantities  of  the  feces  in  salt  solution  is  made  and  1  c.c.  of 
the  filtered  suspension  is  injected  into  the  ear  vein  of  a  rabbit. 
After  a  few  minutes  the  rabbit  is  killed  and  placed  in  the  incubator. 
After  five  hours  of  incubation,  the  rabbit  is  dissected,  and  if  the 
Welch  bacillus  has  been  present  in  the  feces,  small  bubbles  of  gas 
will  have  appeared  in  the  liver  from  which  the  bacilli  may  be  cul- 
tivated in  anaerobic  stab  cultures. 

-Klein,  Ref.  Cent.  f.  Bakt.,  I,  xxx,   1901. 

3  Herter,  <•  '  Common  Bacterial  Infections  of  the  Digestive  Tract, ' '  N.  Y.,  1907. 

*  Welch  and  Nuttal,  Bull.  Johns  Hopkins  Hosp.,  1892,  111,  81. 


212  BIOLOGY  AND  TECHNIQUE 

Bacteriological  examination  of  feces  is  most  often  undertaken 
for  the  isolation  of  Bacillus  typhosus,  dysentery,  cholera,  etc.  These 
methods  are  discussed  in  detail  in  the  chapters  dealing  with  the 
diseases.  See  also  section  on  media. 

The  determination  of  tubercle  bacilli  in  stools  is  difficult  and  of 
questionable  significance,  in  that  they  may  be  present  in  people 
suffering  from  pulmonary  tuberculosis  as  a  consequence  of  swallow- 
ing sputum  or  infected  food,  and  in  that  there  may  be  other  acid- 
bacilli,  such  as  the  timothy  bacillus,  present.  Perhaps  the  most 
reliable  method  is  to  treat  a  suspension  of  the  feces  with  5  per  cent 
antiformin  over  night,  centrifugalize  thoroughly,  wash  the  sediment, 
and  inject  into  guinea  pigs. 

Blood  Cultures. — The  diagnosis  of  septicemia  can  be  positively 
made  during  life  only  by  the  isolation  of  bacteria  from  the  blood. 
Such  examinations  are  of  much  value  and  are  usually  successful 
if  the  technique  is  properly  carried  out.  A  large  number  of  methods 
are  recommended,  the  writers  giving,  however,  only  the  one  which 
they  have  found  successful  and  simple  for  general  use. 

The  blood  is  taken  by  preference  from  the  median  basilic  vein 
of  the  arm.  If,  for  some  reason  (both  forearms  having  been  used 
for  saline  infusion),  these  veins  are  unavailable,  blood  may  be  taken 
from  the  internal  saphenous  vein  as  it  turns  over  the  internal 
malleolus  of  the  ankle  joint.  The  skin  over  the  vein  should  be 
prepared  before  the  specimen  is  taken  by  painting  with  iodin,  as 
for  a  surgical  operation.  The  syringe  which  is  used  should  be  of 
some  sterilizable  variety  (the  most  convenient  the  Luer  model), 
which  is  easily  manipulated  and  does  not  draw  with  a  jerky,  irregular 
motion.  Its  capacity  should  be  at  Jeast  10  c.c.  It  may  be  sterilized 
by  boiling  for  half  an  hour,  or  preferably,  when  all-glass  syringes 
are  used,  they  may  be  inserted  into  potato-tubes  and  sterilized  at 
high  temperature  in  the  hot-air  chamber.  Before  drawing  the  blood, 
a  linen  bandage  is  wound  tightly  about  the  upper  arm  of  the  patient 
in  order  to  cause  the  veins  to  stand  out  prominently.  When  the 
veins  are  plainly  in  view,  the  needle  is  plunged  through  the  skin 
into  the  vein  in  a  direction  parallel  to  the  vessel  and  in  the  direction 
of  the  blood-stream.  After  perforation  of  the  skin,  while  the  needle 
is  groping  for  the  vein,  gentle  suction  may  be  exerted  with  the 
piston.  Great  care  should  be  exercised,  however,  that  the  piston 
is  not  allowed  to  slip  back,  and  air  be,  by  accident,  forced  into  the 
vessel.  In  most  cases  no  suction  is  necessary,  the  pressure  of  the 


BACTERIOLOGICAL  EXAMINATION    OF    MATERIAL  213 

blood  being  sufficent  to  push  up  the  piston.  After  the  blood  has 
been  drawn,  it  should  be  immediately  transferred  to  the  proper  media. 
Epstein  has  recently  recommended  the  mixture  of  the  blood  with 
sterile  two  per  cent  ammonium  oxalate  solution  in  test  tubes,  by 
which  means  the  clotting  is  prevented,  and  transfers  can  be  made 
more  leisurely  to  culture  media.  While  this  method  is  convenient 


FIG.  29. — BLOOD-CULTURE  PLATE  SHOWING  STREPTOCOCCUS  COLONIES.     Note  halo 
of  hemolysis  about  each  colony. 

in  cases  where  blood  must  be  taken  at  some  distance  from  the 
laboratory,  it  is  preferable,  whenever  possible,  to  make  cultures  from 
the  blood  immediately  at  the  bedside. 

The  choice  of  culture  media  for  blood  cultures  should,  to  a 
certain  extent,  be  adapted  to  each  individual  case.  For  routine 
work,  it  is  best  to  employ  glucose  "hormone"  agar  and  glucose-meat- 


214  BIOLOGY  AND  TECHNIQUE 

infusion  broth.  At  least  six  glucose-agar  tubes  should  be  melted 
and  immersed  in  water  at  45°  C.  Before  the  blood  is  mixed  with 
the  medium,  the  agar  should  be  cooled  to  41°  in  order  that  bacteria, 
if  present,  may  not  be  injured  by  the  heat.  The  blood  is  added  to 
the  tubes  in  varying  quantities,  ranging  from  0.25  to  1  c.c.  each, 
in  order  that  different  degrees  of  concentration  may  be  obtained. 
Mixing  is  accomplished  by  the  usual  dipping  and  rotary  motion, 
the  formation  of  air-bubbles  being  thus  avoided.  The  mouth  of 
each  test  tube  should  be  passed  through  the  flame  before  pouring 
the  contents  into  the  plates.  Three  flasks  of  glucose  broth,  contain- 
ing 100  to  150  c.c.  of  fluid  each,  should  be  inoculated  with  varying 
quantities  of  blood — at  least  one  of  the  flasks  containing  the  blood 
in  high  dilution.  The  most  stringent  care  in  the  withdrawal  and 
replacement  of  the  cotton  stoppers  should  be  exercised.5  The  writers 
have  found  it  convenient  to  use,  in  place  of  one  of  these  flasks,  one 
containing,  in  addition  to  the  glucose,  1  gm.  of  powdered  calcium 
carbonate.  This  insures  neutrality,  permitting  pneumococci  or 
streptococci,  which  are  sensitive  to  acid,  to  develop  and  retain  their 
vitality. 

In  making  blood  cultures  from  typhoid  patients,  Buxton  and 
Coleman6  have  obtained  excellent  results  by  the  use  of  pure  ox-bile 
containing  ten  per  cent  of  glycerin  and  two  per  cent  of  pepton  in 
flasks.  The  writers  have  had  110  difficulty  in  obtaining  typhoid 
cultures  by  the  use  of  slightly  acid  meat-extract  broth  in  flasks 
containing  200  or  more  c.c.  to  which  comparatively  little  blood  has 
been  transferred.  The  failure  of  a  proper  blood  culture  service  in 
most  hospitals  is  due,  we  believe,  to  the  fact  that  blood  cultures 
are  taken  by  the  interne  staff,  and  worked  out  by  the  bacteriologist. 
It  is  of  the  utmost  importance,  in  our  opinion,  that  a  single  individual 
should  be  responsible  for  the  entire  examination  from  beginning  to 
end.  This  is  to  avoid  the  great  possibility  of  contamination  in  blood 
culture  work. 

ANAEROBIC  BLOOD  CULTURES. — These  cultures  may  be  taken  by 
mixing  blood  in  deep  tubes  with  glucose-ascitic  agar,  covering  with 
albolene  and  putting  into  Novy  jars. 

In  estimating  the  results  of  a  blood  culture,  the   exclusion  of 


5  Small  Florence  flasks  are  preferable  to  the  Erlenmeyer  flasks  usually  employed, 
^Buxton  and  Coleman,  Am.  Jour,  of  Med.  Sci.,  1907. 


BACTERIOLOGICAL   EXAMINATION    OF    MATERIAL  215 

contamination  usually  offers  little  difficulty.  If  the  same  micro- 
organism appears  in  several  of  the  plates  and  flasks,  if  colonies  upon 
the  plates  are  well  distributed  within  the  center  and  under  the 
surface  of  the  medium,  and  if  the  microorganisms  themselves  belong 
to  species  which  commonly  cause  septicemia,  such  as  streptococcus 
and  pneumococcus,  it  is  usually  safe  to  assume  that  they  emanated 
from  the  patient's  circulation.  When  colonies  are  present  in  one 
plate  or  in  one  flask  only,  when  they  are  situated  only  near  the  edges 
of  a  plate  or  upon  the  surface  of  the  medium,  and  when  they  belong 
to  varieties  which  are  often  found  saprophytic  upon  skin  or  in  air, 
they  must  be  looked  upon  with  suspicion.  It  is  a  good  rule  to  look 
upon  all  staphylococcus  albus  cultures  skeptically. 

Sputum. — In  examining  sputum,  sufficient  emphasis  cannot  be 
placed  upon  the  necessity  of  collecting  the  sputum  in  a  proper  way. 
The  sputum  collected  by  patients  in  the  ordinary  sputum  cup  con- 
sists to  a  very  large  extent  of  material  obtained  from  the  mouth 
and  throat.  If  a  successful  examination  of  sputum  is  to  be  made, 
the  patient  should  be  taught  to  rinse  out  his  mouth  thoroughly,  and 
the  sputum  collected  directly  after  a  cough.  It  is  very  little  to 
ask  for  this  amount  of  care,  if  the  examination  is  really  worth  mak- 
ing at  all.  Sputum  so  collected  should  not  be  left  in  the  ward,  but 
should  be  sent  to  the  laboratory  immediately.  Smears  should  be 
made  on  such  sputum,  with  an  intelligent  idea  of  what  is  desired. 

For  pneumococcus  type  examination,  the  sputum  is  thoroughly 
washed  and  intraperitoneally  injected  into  mice,  according  to  the 
detailed  directions  given  for  typing  in  the  chapter  on  pneumococcus. 

For  influenza  bacillus  examinations,  thin  smears  of  the  sputum 
should  be  stained  by  Gram  and  dilute  carbol  fuchsin.  The  char- 
acteristic grouping  of  influenza  bacilli  is  of  considerable  help.  Plates 
of  chocolate  agar  are  then  streaked. 

Sputum  for  tuberculosis  examination  is  thinly  smeared  and 
stained  by  carbol  fuchsin  or  Hermann's  stain.  When  it  is  desired 
to  carry  the  examination  beyond  this  in  negative  examinations, 
washed  sputum  can  be  injected  into  guinea  pigs,  or  the  sputum  can 
be  antiforminized,  washed  and  then  injected.  Also,  the  anti- 
forminized  sediment  can  be  examined  by  stain  preparation. 

Direct  culture  of  tubercle  bacillus  sputum  can  be  made  by  treat- 
ing with  sodium  hydrate  and  plating  upon  Petroff's  gentian- violet- 
egg  medium,  by  the  method  described  in  detail  in  the  section  on 
tuberculosis. 


216  BIOLOGY  AND  TECHNIQUE 

Throat  Smears  and  Throat  Examinations. — The  bacteriologist,  if 
possible,  should  take  these  specimens  himself,  or  the  physician  tak- 
ing them  should  take  them  only  with  a  clear  illumination  of  the 
throat,  taking  his  specimen  from  the  exact  spot  where  the  lesion  is 
supposed  to  be  located. 

For  diphtheria  examination,  the  specimen  is  taken  with  a 
sterile  swab,  and  plated  directly  upon  Loeffler's  medium.  This 
should  be  incubated  without  delay,  and  the  swab  sent  to  the  labora- 
tory with  the  culture.  The  method  has  been  standardized  and  is 
described  in  the  section  of  diphtheria. 

For  Vincent's  angina  examination,  smears  should  be  taken  and 
stained,  best  by  strong  gentian  violet,  such  as  used  in  the  Gram 
stain,  and  the  smear  searched  for  the  characteristic  spirilla,  and 
fusiform  bacilli.  If  the  patient  is  in  the  laboratory,  it  is  best  to 
make  a  dark  field  examination. 

Examination  of  Lesions  on  the  Genitalia. — Lesions  suspicious  of 
primary  syphilitic  nature  should  be  gently  washed,  the  superficial 
pus  removed,  and  only  exudate  from  the  bottom  of  the  lesion  taken. 
If  necessary,  the  lesion  can  be  gently  scraped  and  serum,  mixed  with 
as  little  blood  as  possible,  used.  No  examination  for  treponema 
pallidum  is  equal  to  the  dark  field  examination.  It  is  important  to 
use  only  thick  slides.  A  drop  of  the  exudate  is  placed  on  the  slide 
and  a  cover-slip  dropped  on  it.  Then  a  drop  of  oil  is  placed  on 
the  bottom  of  the  slide,  over  the  preparation  and  on  the  top  of  the 
cover-slip,  and  the  preparation  is  placed  on  the  dark  field  condenser. 
In  doing  this,  care  should  be  taken  to  avoid  air  bubbles  in  the  oil. 

When  suspicion  of  chancroid  exists,  the  material  should  be  in- 
oculated immediately  into  tubes  of  coagulated  and  inactivated  sterile 
rabbit 's  blood,  and  incubated  according  to  the  method  of  Teague. 

BACTERIA  HABITUALLY  INHABITING  THE  NORMAL  HUMAN  BODY 

In  studying  bacteria  in  disease,  it  is  of  considerable  importance 
to  have  a  clear  idea  of  the  morphological  and  cultural  characteristics 
of  forms  which  are  frequently  encountered  in  different  parts  of  the 
human  body  under  normal  conditions. 

Various  cavities  of  the  body  which  communicate  with  the  ex- 
ternal world,  always  contain  considerable  numbers  of  bacteria  repre- 
senting a  large  variety  of  species.  Some  of  these  may  be  habitual 
saprophytes  associated  with  that  particular  part  of  the  body,  others 


BACTERIOLOGICAL  EXAMINATION   OF   MATERIAL  217 

may  be  accidental  and  temporary  invaders,  members  of  pathogenic 
groups  which,  either  because  of  the  reduced  virulence  of  the  strains 
or  the  increased  resistance  of  the  individual  are  not  capable  under 
the  circumstances  of  causing  their  specific  infection. 

It  is  such  conditions  which  may  lead  to  many  erroneous  etiological 
conclusions  and  which  render  the  investigation  of  the  causation 
of  diseases  in  the  mouth,  intestines  and  other  locations  extremely 
difficult.  It  is  perhaps  best  to  discuss  this  subject  from  the  point 
of  view  of  the  individual  locations  studied. 

Bacteria  in  the  Normal  Mouth  and  Pharynx. — The  mouth  and 
pharynx  are  habitually  the  habitat  of  numerous  bacteria.  Saliva 
itself  is  not  a  good  culture  medium,  and,  indeed,  may,  according 
to  some  investigators,  show  very  slight  inhibitory  or  even  bacteri- 
cidal powers.  But  these,  at  best,  are  not  very  potent,  and  the  saliva 
thus  is  a  basis  for  a  fluid  medium  which  furnishes  water  as  a  solvent 
and  a  reaction  suitable  for  a  great  many  different  bacteria. 

Sloughing  epithelium,  decayed  teeth  and  gums,  food  particles, 
etc.,  furnish  suitable  nutrition.  Catarrhal  inflammation  which  is 
rarely  entirely  absent  to  some  degree  or  in  some  place  in  the  adult 
human  being,  favors  the  lodgment  of  bacteria  upon  the  membranes 
and  reduces  the  resistance  of  the  tissues. 

In  spite  of  these  facts,  it  is  surprising  that  the  frequent  accidental 
injury  of  the  gums  and  oral  and  pharyngeal  mucous  membranes 
so  rarely  leads  to  serious  infection,  and  ends  so  readily.  This  is 
a  fact  which  has  not  as  yet  been  adequately  explained. 

Staphylococci  can  almost  always  be  isolated  from  the  mouth.  They 
are  usually  of  the  Albus  variety,  but  not  infrequently  staphylococcus 
Aureus  also  can  be  found. 

Of  the  streptococci  the  Viridans  is  almost  always  present.  The 
isolation  of  a  "viridans"  from  inflammatory  processes  of  the  mouth 
and  throat,  therefore,  has  very  little  true  significance,  unless  it  is 
isolated  from  a  closed  process,  such  as  a  tooth  abscess,  or  unless  other 
strong  corroborative  evidence  can  be  adduced.  The  Hemolyticus 
variety  is  less  frequently  found  in  the  normal  mouth,  but  may  be 
present  without  causing  disease.  However,  the  isolation  of  a  hemoly- 
ticus  from  an  inflamed  tonsil  or  pharynx  is  much  more  likely  to 
mean  that  there  is  an  etiological  relationship,  and  it  is  of  course  well 
known  that  many  of  the  severe  inflammations  in  this  location  are 
of  hemolyticus  origin. 

In  examinations  made  many  years  ago  by  the  writer,  30  per  cent 


218  BIOLOGY  AND  TECHNIQUE 

of  people  examined  harbored  pneumococci  in  their  mouths,  at  one 
time  or  another,  in  the  course  of  the  cold  months.  Since  then,  the 
typing  of  the  pneumococcus  has  made  it  possible  to  show  that  the 
pneumococci  most  frequently  present  in  the  mouth  belong  to  Group 
IV.  In  the  investigations  of  Dochez  and  Avery  which  are  described 
in  another  place,  it  was  found  that  this  type  caused  only  about  9.8 
per  cent  of  pneumonias,  but  was  found  with  considerable  frequency 
in  normal  mouths.  The  other  and  more  virulent  types  may  be  found 
in  the  normal  mouth,  as  well,  but  are  more  apt  to  represent  recent  con- 
tact with  pneumonia  cases  or  a  transitory  carrier  state.  This,  at  least, 
is  suggested  by  the  writers  mentioned  above,  though  probably  a 
definite,  conclusive  statement  cannot  be  made  concerning  it  at  the 
present  time. 

Of  the  non-pathogenic  Gram-positive  cocci  the  Micrococcus  Candi- 
cans  and  occasional  pigment  forming  micrococci  are  not  infrequent. 

Micrococcus  Tetragenus  is  very  often  an  inhabitant  of  the  mouth 
and,  as  a  matter  of  fact,  one  sees  it  most  frequently  in  routine  work 
in  Loeffler's  cultures  taken  for  the  purpose  of  diphtheria  diagnosis. 

Of  Gram-negative  micrococci  there  is  a  considerable  variety  which, 
without  being  pathogenic,  may  be  cultivated  from  the  mouth  and 
throat  and  add  no  little  confusion  to  meningococcus  carrier  examina- 
tions. Most  common  among  these  are  the  Micrococcus  Catarrlialis, 
which  is  described  in  another  section,  and  may  be  distinguished  from 
the  meningococcus  by  its  heavier  growth,  its  growth  at  room  tem- 
perature and  its  failure  to  produce  fermentation  of  dextrose  and 
maltose.  The  Micrococcus  Flavus,  which  frequently  has  led  to  error 
in  similar  work,  is  a  pigment  forming  Gram-negative  coccus  often 
found  in  the  throat,  which  grows  at  room  temperature,  and  in  most 
cases  agglutinates  spontaneously  in  normal  horse  serum.  Another 
which  forms  very  dry  colonies,  the  Micrococcus  Pkaryngis  siccus,  is 
often  isolated,  but  easily  recognized.  In  addition  to  this,  Elser  and 
Huntoon  have  described  three  different  chromogenic  groups  of  similar 
organisms  often  found  in  the  mouth  and  throat.  These  probably  do 
not  exhaust  all  the  possible  Gram-negative  micrococci  that  can  be 
isolated  from  this  locality,  but  it  is  really  only  of  importance  to  make 
sure  in  human  examination  whether  one  is  dealing  with  a  true 
meningococcus,  with  a  micrococcus  catarrhalis,  or  with  other  sapro- 
phytes. 

True  meningococci  are  of  course  often  found  in  normal  or  slightly 
inflamed  throats  during  the  carrier  state,  which  is  discussed  at  con- 


BACTERIOLOGICAL   EXAMINATION   OF   MATERIAL  219 

siderable  length  in  another  place.  As  discussed  there,  these  organisms 
when  they  are  present  are  usually  located  high  up  in  the  pharynx 
near  its  roof,  and  successful  search  for  carriers  depends  very  largely 
upon  care  of  reaching  the  right  spot  with  the  swab. 

Of  bacilli,  the  mouth  contains  a  large  variety  at  different  times. 
Few  of  these,  however,  are  confusing  from  the  bacteriologist's  point 
of  view,  except  some  of  the  diphtheroids.  The  pseudo-diphtheria 
bacillus,  or  Bacillus  Hoffmanni,  may  be  present  without  having  any 
relationship  to  disease.  It  is  described  in  another  section.  The  other 
larger  and  more  irregular  diphtheroids  are  not  uncommon,  and  are 
easily  distinguished  from  true  diphtheria  bacilli  by  their  appearance 
and  cultural  characteristics. 

Chain- forming  Gram-positive  bacilli  and  large  obviously  sapro- 
phytic  varieties  may  be  present  in  very  dirty  mouths,  but  offer  no 
bacteriological  difficulties. 

Of  the  Gram-negative  bacilli,  Proteus,  Lactis  Aerogenes,  and  special 
members  of  the  Friedldnder  group  may  be  present.  We  have  known 
one  man  who  habitually  had  a  Friedlander  culture  in  his  mouth,  with- 
out ever  suffering  any  harm  from  its  presence. 

The  fusiform  bacillus  described  in  another  section  in  connection 
with  Vincent's  angina,  is  almost  always  present  between  the  gums  and 
the  teeth  in  mouths  that  are  dirty,  with  carious  teeth  or  where  there 
is  some  inflammation  of  the  gums  themselves.  It  is  an  observation 
that  we  make  almost  every  year  with  our  students,  that,  if  a  platinum 
loop  is  passed  between  the  base  of  the  tooth  and  the  gums,  and  smears 
taken  from  a  number  of  students,  the  bacteria  usually  associated  with 
Vincent's  angina,  spirochsetes  and  fusiform  bacilli,  can  be  seen  in 
one  or  another  of  the  cases  examined. 

Spirilla  and  spirochaetes  are  almost  habitually  present.  The 
Spirillum  Milleri,  named  after  Miller,  who  has  made  valuable  studies 
upon  mouth  bacteria,  is  a  small  true  spirillum,  easily  cultivated,  and 
not  easily  confused  with  other  morphologically  similar  organisms. 
Miller  cultivated  three  of  four  varieties  of  mouth  spirilla. 

True  treponema  (Noguchi's  classification),  are  almost  always 
present  in  locations  like  those  described  for  the  fusiform  bacilli,  and 
even  on  the  mucous  membranes,  especially  when  small  spots  of  necrosis 
or  inflammation  occur.  Most  frequently  discussed  among  these  are 
the  large  spirochaete,  associated  with  Vincent's  angina,  the  Spironema 
Vincenti.  There  are,  likewise,  present  very  frequently  the  treponema 
macrodentium  and  microdentium,  classified  thus  by  Noguchi.  These 


220  BIOLOGY  AND  TECHNIQUE 

organisms  are  best  observed  under  the  dark  field,  but  can  also  be 
stained  in  smear  if  strong  gentian  violet  or  carbol  fuchsin  are  used. 
It  is  important  to  note  that  morphologically  the  macrodentium  is  very 
similar  to  the  treponema  pallidum,  and  in  the  dark  field  examination 
of  syphilitic  lesions  of  the  mouth  and  throat,  this  similarity  must  be 
carefully  taken  into  account.  We  have  seen  cases  in  which  we  were 
unwilling  to  make  a  definite  diagnosis  on  these  findings  alone.  It 
is  our  belief  that  whenever  extensive  necrosis  of  the  tissues  of  the 
mouth  and  pharynx  occur  in  consequence  of  other  infection  or  or  injury, 
the  necrotic  tissues  are  apt  to  be  invaded  by  fusiform  bacilli,  and 
spirochaetes,  which  in  subsequent  examination  dominate  the  bac- 
teriological picture.  We  believe,  however,  that  in  the  large  majority 
of  these  cases,  perhaps  including  the  clinical  picture  spoken  of  as 
Vincent's  angina,  the  trepdnemata  and  fusiform  bacilli  are  secondary 
to  the  primary  etiological  factors,  such  as  those  mentioned.  These 
organisms  are  anaerobic.  We  believe  that  the  early  contention  of 
Tunnicliff  that  the  spirochaetes  and  fusiform  bacilli  found  in  Yin- 
cent  's  Angina  are  different  stages  of  the  same  organism,  is  not  generally 
accepted  to-day. 

The  normal  mouth  is  also  apt  to  contain  occasional  members  of 
the  Leptotkrix  and  Streptothrix  groups.  One  of  these,  the  LeptotJirix 
innominata  of  Miller,  is  supposed  to  be  characteristic  of  the  mouth 
flora.  It  may  appear  as  a  large  Gram-positive  bacillus  form  which 
is  believed  by  some  writers  to  be  a  true  bacillus,  rather  than  a  lepto- 
thrix,  and  is  spoken  of  as  the  Bacillus  Maximus  Buccalis  (Miller). 

Bacteria  in  the  Nose  and  Accessory  Sinuses. — That  the  nasal  mucosa 
should  be  a  favorable  site  for  the  deposit  of  numerous  microorgan- 
isms follows  from  the  fact  that  air  is  constantly  passing  in  and  out 
during  respiration.  The  varieties  of  bacteria  to  be  found  in  the 
nose,  therefore,  may  belong  to  any  that  happen  to  be  present  in 
the  inhaled  air. 

The  subject  of  the  bacteriology  of  the  nose  deserves  more  atten- 
tion than  has  been  given  to  it  during  recent  years,  for  infections 
of  the  nasal  sinuses  and  the  conditions  which  lead  to  them,  are  being 
recognized  of  the  utmost  importance  upon  general  health.  Earlier 
investigators  claimed  that  the  passage  of  bacteria  in  the  air  to  the 
deeper  respiratory  organs  is  very  largely  arrested  by  a  sort  of 
filtering  action  in  the  nose.  Thomson  and  Hewlett7  found  that  in 

''Thomson  and  Hewlett,  Baumgarten's  Jahresb.,   12,  1896,  767. 


BACTERIOLOGICAL   EXAMINATION    OF    MATERIAL  221 

animals,  the  tracheal  mucus,  as  well  as  the  mucous  membrane  of  the 
posterior  portions  of  the  healthy  nose,  are  usually  sterile,  although 
the  vestibulium  nasae  is  usually  heavily  contaminated.  When  the 
nasal  cavity  and  the  septum  were  artificially  inoculated  with  Bacil- 
lus prodigiosus,  the  organisms  disappeared  in  about  two  hours. 
These  observers  believed  that  the  healthy  nasal  mucus  is  not  bac- 
tericidal, but  does  not  favor  growth.  They  examined  air  which 
passed  through  the  nose,  and  found  that  in  the  case  of  air  which  con- 
tained over  20  mould  spores  and  9  bacteria  per  cubic  centimeter,  these 
organisms  almost  entirely  disappeared  in  the  passage  of  the  air  through 
the  nose.  Hilderbrandt8  has  previously  obtained  similar  results  in 
Baumgarten's  laboratory.  Wright9  also  has  made  similar  investiga- 
tions, and  showed  that  between  3/4  to  4/5  of  the  bacterial  flora  of 
the  inspired  air  was  held  back  in  its  passage  through  the  nose. 

Among  the  most  interesting  studies  along  these  lines  are  those 
of  Neumann10  who  studied  the  nasal  secretions  of  over  200  people, 
of  which  about  111  were  supposedly  normal.  Neumann  found  in 
normal  noses  a  very  large  number  of  different  microorganisms.  The 
percentage  findings  of  various  bacteria  were  as  follows: 

Pseudo-diphtheria  (probably  including  diphtheroids) — 98  to  100  per  cent 

Micrococcus  albus — 98  per  cent 

Micrococcus  aureus — 30  per  cent 

Streptococcus  lancelatous   (probably  pneumococcus) — 4  per  cent 

Friedlander  bacilli — 6  per  cent 

Micrococcus  citreus — 12  per  cent 

Colon  bacilli — 12  per  cent 

Streptococcus — 2  per  cent 

Molds — 20  per  cent 

Sarcinae — 6  per  cent 

Lactis  aerogenes — 4  per  cent 

Yeasts — 2  per  cent 

Neumann  mentions  other  microorganisms  in  addition  to  these, 
but  the  figures  given  are  sufficient  to  show  that  the  normal  nose 
may  contain  almost  any  of  the  known  organisms  including  a  great 
many  of  the  non-pathogenic  forms  in  air,  of  which  the  bacteriologist 
dealing  with  disease  knows  very  little  as  a  rule.  Calamida  and 


*  Hilderbrandt,  Baumgarten's  Jahresb.,  4,   1888,  378. 
9  Wright,  quoted  from  Baumgarten  's  Jahresb.,  5,  1889. 
"Neumann,  Zeit.  f.  Hyg,,  40,   1902,  33. 


222  BIOLOGY  AND  TECHNIQUE 

Bertarclli11  also  carried  out  interesting  studies  on  the  normal  bac- 
terial flora  of  the  accessory  nasal  sinuses.  Working  first  with  dogs, 
they  found  that  in  20  dogs  of  various  ages,  the  frontal  sinuses  were 
always  sterile.  In  a  single  case  they  isolated  an  organism  which 
resembled  the  Colon  bacillus. 

In  8  dogs  the  ethmoidal  sinuses  were  sterile.  In.  16  of  20  dogs 
the  maxillary  sinuses,  antrum  of  Highmore,  were  sterile.  In  the 
others  they  found  various  cocci.  When  they  inoculated  the  naso- 
pharynx of  dogs  with  cultures  of  B.  prodigiosus,  pyocyaneus,  and 
subtilis,  and  killed  them  8  to  24  hours  later,  3  of  them  showed 
entirely  sterile  accessory  sinuses  and  sterile  middle  ear.  One  animal 
gave  a  positive  culture  of  prodigiosus  in  the  antrum,  frontal  sinuses, 
and  the  ear.  In  3  others,  only  the  antrum  was  infected.  Two  of 
the  animals  treated  with  B.  pyocyaneus  retained  sterile  sinuses.  Of 
6  treated  with  Subtilis,  4  were  entirely  sterile.  It  is  interesting  to 
note  that  the  animals  that  were  infected,  were  those  which  were 
killed  only  8  to  10  hours  after  inoculation.  Of  those  which  were 
killed  between  18  and  24  hours  after  inoculation,  all  but  one  were 
sterile.  They  examined  12  fresh  human  cadavers  within  a  few  hours 
after  death,  never  later  than  20  hours.  In  all  but  one  of  these 
all  accessory  sinuses  of  the  nose  were  sterile,  and  in  this  one  a  non- 
pathogenic  Staphylococcus  Albus  was  found.  Kuster,  who  has  sum- 
marized work  on  the  nasal  flora  in  the  Kolle  and  Wassermann,  on 
the  basis  of  a  study  of  the  literature  as  well  as  his  own  investiga- 
tions, comes  to  the  conclusion  that  we  cannot  speak  of  a  character- 
istic nasal  flora,  that  practically  all  of  the  organisms  with  which 
man  can  come  in  contact  through  the  air  may  settle  there  for  a 
shorter  or  longer  period.  In  a  healthy  nose,  however,  few  organisms 
can  gain  a  permanent  foothold,  largely  because  of  unsuitable  cultural 
conditions  and  of  the  action  of  leucocytes  and  the  secretions. 

Bacteria  in  the  Tissues  Themselves. — Recent  work  has  given  definite 
evidence  that  even  the  tissues  themselves  may  not  be  sterile  in 
normal  human  beings.  This  has  led,  •  we  believe,  to  a  certain 
amount  of  error  in  etiological  conclusions  when  blood  cultures  and 
cultures  from  normal  or  slightly  diseased  lymphatic  tissues  have 
been  taken,  and  diphtheroid  and  various  coccus  forms  isolated. 
According  to  the  experiments  done  by  Adami12  there  is  a  constant 

"Calamida  and  Bertarelli,  Ziet.  f.  Bakt.,  I  Orig.,  32,  1902, 
*- Adami,  Jour.  A.  M.  A.,  Dec.,  1899, 


BACTERIOLOGICAL   EXAMINATION    OF    MATERIAL  223 

entrance  of  bacteria  into  the  portal  circulation  from  the  intestines. 
These  are  very  largely  disposed  of  in  the  liver,  but  it  may  well  be 
that  the  liver  does  not  always  eliminate  all  the  bacteria  from  the 
portal  circulation,  and  that  some  of  these  then  lodge  in  other  tissues 
and  become  latent  there. 

The  latency  of  bacteria  in  the  healthy  body  can  no  longer  be 
questioned.  We  have  long  known  that  treponema  pallidum  the 
spirochsetes  that  infect  mice,  and  many  trypanisomes  can  remain 
present  for  a  long  time  in  the  circulation  and  in  the  tissues  of  animals 
and  man  without  giving  rise  to  characteristic  symptoms  or  even 
to  any  symptoms.  We  have,  ourselves,  found  pallida  in  the  testes 
of  rabbits  four  months  after  inoculation  without  there  having  been 
the  slightest  tissue  reaction,  and  in  human  syphilis  this  latency  is 
well  recognized.  That  tetanus  spores  may  remain  latent  in  the 
spleen  and  other  organs  of  guinea  pigs  under  certain  experimental 
conditions,  has  been  shown  by  the  Italian  observer,  Canfora,13  and 
recently  we  have  seen  a  very  convincing  example  of  latency  of 
streptococci  in  the  tissues  of  the  hand.  A  very  severe  hemolytic 
streptococcus  lesion  subsided  under  surgical  treatment,  and  four 
months  later  a  purely  cosmetic  secondary  operation  was  undertaken 
at  a  time  when  there  was  not  the  slightest  trace  of  infection,  and 
hemolytic  streptococci  were  again  isolated  from  the  tissues  at  this 
operation.  There  was,  incidentally,  no  sign  of  infection  of  the 
wound,  which  healed  uneventfully. 

The  investigations  of  Torrey  and  others  have  shown  that  from 
lymph  nodes,  the  seat  of  various  non-bacterial  conditions,  such  as 
sarcoma,  Hodgkin's  disease,  etc.,  many  varieties  of  diphtheroids  may 
be  isolated,  and  Roscnau  has  reported  a  number  of  blood  culture 
results  in  which  diphtheroids  and  cocci  were  isolated  from  the  blood 
in  the  presence  of  febrile  conditions  which  obviously  were  not  due 
to  the  particular  organisms  isolated. 

Not  much  can  be  said  about  this  problem  of  latency  at  the  present 
time  because  little  is  known  about  it,  but  the  possibility  should  be 
kept  in  mind,  and  should  cause  great  conservatism  whenever  isola- 
tions from  the  tissues  are  made,  and  the  etiological  question  is  raised. 

The  Bacteriology  of  the  Intestinal  Tract. — More  than  any  other 
part  of  the  body,  the  intestinal  canal  has  a  specific  flora  of  its  own. 
This  varies  at  different  ages,  with  health  and  disease,  and  is  to 

"Can-fora,  Cent.  f.  Bakt.,  45,  1908. 


224  BIOLOGY  AND  TECHNIQUE 

a  considerable  extent  dependent  upon  diet.  Also,  many  of  the  bac- 
teria that  cause  specific  diseases  of  the  intestinal  canal,  such,  for 
instance,  as  the  typhoid  bacillus,  the  paratyphoid  bacilli,  the  dysen- 
tery group,  and  some  of  the  doubtfully  pathogenic  organisms  like 
the  Morgan  bacilli,  are  very  closely  related  in  morphology  and  cul- 
tural reactions  to  non-pathogenic  and  saprophytic  inhabitants  of 
the  bowel.  In  no  type  of  bacteriological  work,  therefore,  is  it  more 
necessary  to  have  an  intelligent  understanding  of  the  bacterial 
species  that  are  likely  to  be  found  without  pathogenic  significance. 

Furthermore,  the  intestinal  canal  is  a  large  test  tube  from  which 
bacterial  products  can  be  absorbed  in  sufficient  amounts  to  cause 
severe  illness.  In  it,  different  kinds  of  food  supply  nutritive  ma- 
terial which  may  favor  one  or  another  species,  and  various  condi- 
tions of  aerobiosis  and  anaerobiosis  may  prevail.  It  is  more  than 
likely,  therefore,  that  many  so-called  cases  of  intestinal  poisoning, 
formerly  loosely  spoken  of  as  ptomain  poisoning,  may  be  caused 
by  substances  formed  within  the  intestine  by  bacterial  action  upon 
the  food,  rather  than  upon  the  relatively  smaller  amount  of  fermen- 
tative and  putrefactive  products  taken  in. with  partially  decomposed 
food. 

The  intestinal  canal  of  the  child  at  birth  is  sterile.  The  meconium 
of  such  children  has  been  found  by  many  investigators  to  be  free 
from  bacteria.  But  this  does  not  last  very  long.  Within  a  few 
hours  after  birth,  infection  takes  place,  and  from  then  until  death, 
the  intestinal  canal  is  constantly  the  seat  of  a  voluminous  and  varied 
bacterial  life.  Kendall,14  who  has  written  much  on  this  subject, 
and  in  his  book  has  brought  together  much  of  the  information, 
gathered  from  the  researches  of  Escherich,15  Herter,10  and  his  own 
investigations,  has  classified  the  different  stages  of  the  bacterial  flora 
in  man,  as  follows: 

1.  Bowel  at  birth,  sterile. 

2.  First  to  the  third  day  a  period  of  "  adventitious  bacterial  in- 
fection.'' 

After  this  time  there  is  the  period  of  establishment  of  the  charac- 
teristic infantile  intestinal  flora  which  gradually  changes  as  the  diet 

"Kendall,  Bacteriology,  General,  Pathological  and  Intestinal,  Lea  &  Febiger, 
Phila.,  1916,  p.  580. 

15  Escherich,  Darmbakterien  des  Sauglings,  Stuttgart,  1886,  p.  9. 

18  Herter,  The  Common  Bacterial  Infections  of  the  Digestive  Tract,  Harvey 
Lect.,  1906-1907. 


BACTERIOLOGICAL   EXAMINATION    OF    MATERIAL  225 

approaches  more  and  more  that  of  the  adult,  into  the  characteristic 
flora  of  the  adult. 

In  the  earliest  days  during  the  stage  of  "adventitious  infection/' 
when  the  child  is  getting  its  first  bacteria  from  the  air  and  subjects 
with  which  its  mouth  comes  in  contact,  the  bacterial  flora  is  de- 
termined largely  by  accident. 

When  the  child  begin  to  take  food,  it  is  of  great  importance 
for  the  determination  of  the  bacterial  flora,  whether  it  is  being 
breast  fed  or  being  fed  on  artificially  modified  cows '  milk. 

In  breast  fed  children,  the  upper  part  of  the  small  intestine  will 
usually  contain  enterococcus,  streptococcus  lacticus,  and  a  general 
predominance  of  the  coccoid  form.  Lower  down  toward  and  behind 
the  ileocecal  valves,  the  B.  lactis  aerogenes  and  the  Colon  bacilli 
appear.  In  the  lower  parts  of  the  cecum  and  the  rectum,  the  anaero- 
bic Bacillus  bifidus  of  Tissier  and  similar  anaerobes  predominate, 
and  many  proteolytic  bacteria  may  be  present. 

In  contrast  to  this,  in  artificially  fed  infants,  the  bowel  is  rela- 
tively richer  in  the  Colon  group,  and  the  B.  aerogenes  type;  B. 
mesentericus  and  other  anaerobic  spore  formers  will  be  present  in 
considerable  numbers,  and  in  the  lower  bowel  the  B.  bifidus  types 
are  largely  replaced  by  Colon  bacilli,  B.  acidophilus  and  similar 
organisms.  Very  early  there  may  be  also  present  in  children  a 
curious  little  tetanus-like  organism  spoken  of  as  Bienstock's  B. 
putrificus. 

Tissier,  who  has  done  a  great  deal  of  work  on  this  problem, 
described  the  flora  of  a  five  year-old  child  in  which  the  gradual 
transition  from  the  milk  to  the  mixed  diet  was  taking  place  as 
follows.  We  quote  from  Kuster.17  "Constant  fundamental  flora, 
B.  bifidus,  enterococcus,  Colon  bacillus,  B.  acidophilus.  Variable 
adventitious  organisms,  B.  perfringens,  cocci,  and  a  number  of  other 
Gram-negative  bacilli,  together  with  some  yeasts.'* 

As  adult  life  is  attained,  there  is  a  gradual  relative  increase 
of  organisms  of  the  Colon  type,  which  eventually  constitute  about 
75  per  cent  of  the  intestinal  bacteria. 

In  the  normal  adult  the  stomach  is  usually  sterile.  The  duodenum 
contains  a  few  cocci  and  Gram-positive  and  negative  bacilli,  not  of 
the  Colon  type.  There  is  a  gradual  numerical  increase  of  bacteria 
downward.  In  the  jejunum,  or  upper  ileum,  the  Colon  types  begin 


17  Kuster,  Kolle  and  Wvssermann,  2nd,  Edition,  Vol.  6,  p.  469. 


226  BIOLOGY  AND  TECHNIQUE 

to  grow  numerous,  and  in  the  cecum  and  colon,  which  are  the  seat 
of  the  greatest  bacterial  activity,  the  flora  consist  chiefly  of  Colon 
types,  B.  mesentericus,  a  few  anaerobic  spore  formers  of  the  Welch 
bacillus  type,  and  Gram-positive  cocci. 

All  who  have  studied  this  subject  have  found  that  diet  has  a 
definite  and  important  bearing  upon  the  intestinal  flora  and  that 
definite  changes  may  be  brought  about  in  the  bacterial  contents 
of  the  bowel  by  purposefully  adjusting  the  diet.  The  studies  of 
Herter16  and  of  Kendall/4  particularly,  have  been  contributed  to 
our  knowledge  of  this  subject.  Herter  has  laid  particular  stress 
upon  the  importance  of  the  Welch  bacillus  and  its  subvarieties  upon 
intestinal  putrefaction.  In  this  he  is  not  entirely  in  agreement  with 
Eettger18  and  others  who  believe  that  the  Welch  bacillus  attacks 
proteins  but  slightly,  being  chiefly  concerned  with  carbohydrate 
fermentation.  Herter  has  produced  indicanuria  in  dogs  by  feeding 
large  amounts  of  meat,  and  found  that  with  such  feeding  the  colon 
and  ileum  contained  considerable  numbers  of  anaerobic  bacilli.  He 
also  believes  that  this  bacillus  is  particularly  concerned  with  a 
chronic  putrefactive  activity  which  takes  place  in  the  large  intes- 
tine, in  the  course  of  which  anaerobic  bacilli  produce  butyric  acid. 
In  consequence  of  this,  there  may  be  a  considerable  intestinal  irrita- 
tion and  carbohydrate  intolerance.  Considerable  anuria  may  also 
be  a  consequence.  Other  writers  like  Friedman19  believe  that  con- 
stipation favors  the  increase  of  these  putrefactive  organisms. 
Simonds20  has  made  an  exhaustive  study  of  the  relationship  of  the 
Welch  bacillus  group  to  intestinal  conditions,  and  has  reviewed  the 
literature  extensively.  He  summarizes  his  studies  on  this  problem 
as  follows:  "In  the  case  of  gas  bacillus  diarrhea,  the  presence  of 
an  excess  of  carbohydrates  in  the  intestinal  content  brings  about 
conditions  in  the  lower  ileum  and  first  part  of  the  colon  which  are 
particularly  conducive  to  the  growth  of  B.  Welchii.  The  absence 
of  lactic  acid  producing  bacteria,  as  pointed  out  by  Kendall,  renders 
conditions  still  more  favorable  to  the  multiplication  of  these  or- 
ganisms. They,  therefore,  rapidly  increase  in  numbers,  produce 
irritating  butyric  acid,  and  are  swept  on  in  excessive  numbers  into 
the  lower  bowel.  The  number  of  spores  produced  will  be  measurably 

18  Eettger,  Jour,  of  Biol.  Chem.,  2,  1906,  71. 

10  Friedman,  Transac.  of  Chicago  Pathol.  Soc.,  1901,  cited  from  Simonds. 

20  Simonds,  Monograph  of  Rock.  Institute,  No.  5,  1915. 


BACTERIOLOGICAL   EXAMINATION    OF    MATERIAL  227 

proportional  to  the  number  of  bacilli  which  reach  the  lower  part 
of  the  bowel;  hence,  the  excessive  number  of  spores  "of  Bacillus 
Welchii  in  the  stools  in  cases  of  gas  bacillus  diarrhea."  Simonds' 
results  substantiate  the  work  of  Kendall  and  Day21  to  the  effect 
that  children  and  adults  with  diarrhea  who  showed  large  numbers 
of0  gas  bacilli  in  the  stools  are  made  worse  by  feeding  sugars,  and 
that  prompt  improvement  results  when  the  diet  is  changed  to  one 
largely  composed  of  protein.  An  absence  of  lactic  acid  by  the  feed- 
ing of  butter-milk  still  further  aids  in  eliminating  the  Welch  bacillus. 
Kendall  has  shown  by  prolonged  experimentation  on  monkeys,  dogs 
and  cats  that  feeding  with  cows'  milk,  to  which  sufficient  lactose 
has  been  added  to  simulate  breast  milk,  produces  a  bacterial  flora 
in  such  animals  which  approaches  that  of  the  normal  nursing  infant. 
The  stools  take  on  an  acid  reaction,  and  organisms  like  B.  bifidus 
and  the  Enterococcus  begin  to  predominate.  In  order  to  bring  this 
about,  he  states,  it  is  necessary  to  continue  the  feeding  for  consider- 
able periods.  Kendall  divides  the  pathological  cases  in  which  it 
can  be  reasonably  suspected  that  abnormal  bacterial  conditions  of 
the  intestinal  tract  play  a  causative  part,  into  those  which  are  due 
to  the  action  of  the  bacteria  upon  proteins,  and  those  in  which  it 
is  chiefly  a  matter  of  carbohydrate  fermentation.  In  the  case  of 
the  abnormal  proteolytic  processes,  there  may  be  a  liberation  of 
substances  like  histamin  and  other  toxic  amines,  and  there  may 
even  be  the  formation  of  specific  toxins  such  as  those  which  have 
been  recently  produced  by  Bull  and  others  from  the  Welch  bacillus. 
Abnormal  carbohydrate  splitting'  may  result  in  hyperacidity  and 
in  stasis  of  the  bowel,  and  secondary  putrefaction  in  consequence 
of  this. 

It  is  due  to  studies  like  those  of  the  writers  mentioned  above 
that  we  may  hope  to  be  able  to  exert  considerable  therapeutic  in- 
fluence upon  abnormal  intestinal  conditions  by  altering  the  flora  of 
the  intestine,  on  the  one  hand  by  controlling  the  diet,  and  on  the 
other  hand  by  inoculating  with,  or,  in  other  words,  feeding  bacteria 
of  a  type  which  may  correct  the  condition  that  exists. 

A  detailed  study  of  these  therapeutic  measures  cannot  be  given 
in  tliis  place.  They  are  treated  of  in  articles  like  those  of  Coleman 
and  Shaffer,22  in  Kendall's  book  from  which  we  have  quoted  freely, 


21  Kendall  and-  Day,  Boston  Med.  and  Surg.  Jour.,  1911,  741  and  1912,  753. 
"Coleman  and  Shaffer,  Archiv.  Inter.  Med.,  Vol.  4,   1909. 


228  BIOLOGY  AND  TECHNIQUE 

and  in  the  publications  of  Herter,  Kendall,  Rettger,  Torrey23  and 
others. 


BACTERIA  IMPORTANT  BECAUSE  OF  THEIR  FREQUENT  PRESENCE 
IN  THE  INTESTINAL  CHANNEL 

Bacillus  Acidophilus.2* — A  Gram-positive  bacillus  which  easily 
undergoes  granular  degeneration  and  grows  under  conditions  of 
acidity,  not  supported  by  most  other  bacteria.  It  is  closely  related 
to  the  Bacillus  bulgaricus,  does  not  form  spores,  and  is  closely 
related  to  a  group  of  similar  organisms  which  have  not  been  suffi- 
ciently studied  to  be  conclusively  classified.  (See  Kendall,  Jour. 
Med.  Res.,  1910,  22,  and  Rahe,  Jour.  Infec.  Dis.,  15,  1914,  41.)  Its 
isolation  can,  according  to  Kendall,  best  be  accomplished  by  in- 
oculating the  original  material  into  dextrose  broth  containing  0.25 
per  cent  acetic  acid.  Two  or  three  transfers  on  a  medium  like  this 
after  intervals  of  several  days  will  give  pure  cultures.  Does  not 
liquefy  gelatin,  has  been  associated  by  Escherich  with  acute  diarrhea 
in  children.  Does  not  produce  gas. 

Bacillus  AcidopMlus  Aerogenes. — Described  by  Torrey  and  Rahe25 
which  closely  resembles  the  Bacillus  acidophilus,  except  that  it 
produces  gas  in  mono-,  di-  and  some  polysaccharids. 

Bacillus  Bifidus  of  Tissier. — This  is  a  strictly  anaerobic  bacillus. 
It  was  described  by  Tissier  in  1900.  (See  also,  Noguchi,  Jour. 
Exper.  Med.,  12,  1910,  182.)  It  is  spoken  of  as  Bifidus  because  it 
will  often  show  a  bifid  branching  at  the  ends,  a  condition  in  which 
it  is  not  shown  in  smears  from  the  intestinal  contents.  It  is  found 
early  in  the  stools  of  nursing  children.  It  produces  considerable 
amounts  of  acid,  but  no  gas  from  carbohydrates.  Morphologically 
this  organism  is  often  described  as  Gram-positive,  but,  as  a  matter 
of  fact,  many  members  are  Gram-negative,  and  often  the  bacilli 
themselves  may  be  Gram-negative  with  Gram-positive  granules  scat- 
tered through  them. 

Bacillus  Mesentericus. — This  is  a  Gram-positive,  aerobic,  spore 
bearing  bacillus  which  is  active  proteolytic  and  concerned  with 
putrefaction  in  the  intestinal  channel.  The  organism  is  more  closely 


83  Torrey,  Jour,  of  Infec.  Dis.,  16,  1915,  72. 

84  Moro,  Wien.  klin.  Woch.,  5,  1900 ;  Finkelstein,  Deut.  med..  Woch.,  22,  1900. 
25  Torrey  and  Rahe,  Jour,  of  Infec.  Dis.,  17,  1915,  437, 


BACTERIOLOGICAL   EXAMINATION    OF    MATERIAL  229 

related  to  the  common  hay  bacillus  or  Subtilis.  It  is  actively  motile, 
forms  spores  and  grows  with  great  ease  on  the  simplest  media.  It 
differs  from  the  ordinary  Subtilis  bacillus  in  that  it  does  not  ferment 
dextrose.  Kendall26  has  shown  that  symbiosis  of  the  B.  Mesentericus 
with  the  Colon  bacillus  in  milk  will  produce  a  greatly  increased 
metabolism  of  both. 

Bacillus  Putrificus  of  Bienstock.27 — This  is  a  Gram-positive 
anaerobic  bacillus  which  forms  end  spores,  and,  therefore,  mor- 
phologically resembles  the  tetanus  bacillus.  It  is  identical  probably 
with  Klein's  bacillus  cadaveris  sporogenes.  It  is  capable  of  produc- 
ing powerful  proteolytic  cleavage  in  milk,  cheese,  etc.  Bienstock 
believes  that  it  is  present  constantly  in  normal  feces. 


88  Kendall,  Boston  Med.  and  Surg.  Jour.,  163,  1910. 

27  Bienstock,  Archiv.  f.  Hyg.,  36,  335,  1899,  and  39,  390,  1901. 


SECTION  II 

INFECTION  AND  IMMUNITY 


CHAPTER   XI 

FUNDAMENTAL   FACTOKS   OF   PATHOGENICITY   AND   INFECTION 

WHEN  microorganisms  gain  entrance  to  the  animal  or  human 
body  and  give  rise  to  disease,  the  process  is  spoken  of  as  infection. 

Bacteria  are  ever  present  in  the  environment  of  animals  and 
human  beings  and  some  find  constant  lodgment  on  various  parts  of 
the  body.  The  mouth,  the  nasal  passages,  the  skin,  the  upper  respira- 
tory tract,  the  conjunctivas,  the  ducts  of  the  genital  system,  and 
the  intestines  are  invariably  inhabited  by  numerous  species  of  bac- 
teria, which,  while  subject  to  no  absolute  constancy,  conform  to 
more  or  less  definite  characteristics  of  species  distribution  for  each 
locality.  Thus  the  colon  organisms  are  invariably  present  in  the 
normal  bowel,  Doderlein's  bacillus  in  the  vagina,  Bacillus  xerosis 
in  many  normal  conjunctivae,  and  staphylococcus,  streptococcus, 
various  spirilla,  and  pneumococcus  in  the  mouth.  In  contact,  there- 
fore, with  the  bodies  of  animals  and  man,  there  is  a  large  flora  of 
microorganisms,  some  as  constant  parasites,  others  as  transient  in- 
vaders ;  some  harmless  saprophytes  and  others  capable  of  becoming 
pathogenic.  It  is  evident,  therefore,  that  the  production  of  an  infec- 
tion must  depend  upon  other  influences  than  the  mere  presence  of 
the  microorganisms  and  their  contact  with  the  body,  and  that  the 
occurrence  of  the  reaction — for  the  phenomena  of  infection  are  in 
truth  reactions  between  the  germ  and  the  body  defenses — is  governed 
by  a  number  of  important  secondary  factors. 

In  order  to  cause  infection,  it  is  necessary  that  the  bacteria  shall 
gain  entrance  to  the  body  by  a  path  adapted  to  their  own  respective 
cultural  requirements,  and  shall  be  permitted  to  proliferate  after 
gaining  a  foothold.  Some  of  the  bacteria  then  cause  disease  by 

230 


FACTORS  OF  PATHOGENICITY  AND  INFECTION  231 

rapid  multiplication,  progressively  invading  more  and  more  exten- 
sive areas  of  the  animal  tissues,  while  others  may  remain  localized 
at  the  point  of  invasion  and  exert  their  harmful  action  chiefly  by 
local  growth  and  the  elaboration  of  specific  poisons. 

The  inciting  or  inhibiting  factors  which  permit  or  prohibit  an 
infection  are  dependent  in  part  upon  the  nature  of  the  invading 
germ  and  in  part  upon  the  conditions  of  the  defensive  mechanism 
of  the  subject  attacked. 

Bacteria  are  roughly  divided  into  two  classes,  saprophytes  and 
parasites.  The  saprophytes  are  those  bacteria  which  thrive  best  on 
dead  organic  matter  and  fulfill  the  enormously  important  function 
in  nature  of  reducing  by  their  physiological  activities  the  excreta 
and  dead  bodies  of  more  highly  organized  forms  into  those  simple 
chemical  substances  which  may  again  be  utilized  by  the  plants  in 
their  constructive  processes.  The  saprophytes,  thus,  are  of  extreme 
importance  in  maintaining  the  chemical  balance  between  the  animal 
and  plant  kingdoms.  Parasites,  on  the  other  hand,  find  the  most 
favorable  conditions  for  their  development  upon  the  living  bodies 
of  higher  forms. 

While  a  strict  separation  of  the  two  divisions  cannot  be  made, 
numerous  species  forming  transitions  between  the  two,  it  may  be 
said  that  the  latter,  class  comprises  most  of  the  so-called  pathogenic 
or  disease-producing  bacteria.  Strict  saprophytes  may  cause  dis- 
ease, but  only  in  cases  where  other  factors  have  brought  about  the 
death  of  some  part  of  the  tissues,  and  the  bacteria  invade  the 
necrotic  areas  and  break  down  the  proteins  into  poisonous  chemical 
substances  such  as  ptomains,  or  through  their  own  destruction  give 
rise  to  the  liberation  of  toxic  constituents  of  their  bodies.  It  Is 
necessary,  therefore,  that  bacteria,  in  order  to  incite  disease,  should 
belong  strictly  or  facultatively  to  the  class  known  as  parasitic.  It 
must  not  be  forgotten,  however,  that  the  terms  are  relative,  and  that 
bacteria  ordinarily  saprophytic  may  develop  parasitic  and  patho- 
genic powers  when  the  resisting  forces  of  the  invaded  subject  are 
reduced  to  a  minimum  by  chronic  constitutional  disease  or  other 
causes. 

Organisms  that  are  parasitic,  however,  are  not  necessarily  patho- 
genic, and  there  are  certain  more  or  loss  fundamental  requirements 
which  experience  has  taught  us  must  be  met  by  an  organism  in 
order  that  it  may  be  infectious  (or  pathogenic)  for  any  given 
animal;  and  by  infectiousness  is  meant  the  ability  of  an  organism 


232  INFECTION  AND  IMMUNITY 

to  live  and  multiply  in  the  animal  fluids  and  tissues.  For  instance, 
an  organism  which  is  shown  not  'to  grow  at  the  body  temperature 
•of  warm-blooded  animals  may  safely  be  assumed  not  to  be  infectious 
for  such  animals;  and  experience  is  gradually  teaching  us  that 
strictly  aerobic  organisms,  those  thriving  only  in  the  presence  of 
free  oxygen  and  not  able  to  obtain  this  gas  in  available  combination 
from  carbohydrates,  can  also  be  safely  excluded  from  the  infectious 
class.  We  have  also  learned  that  anaerobic  organisms,  although 
infectious  when  gaining  entrance  to  tissues  not  abundantly  supplied 
with  blood,  are  practically  unable  to  multiply  in  the  blood  stream 
and  give  rise  to  generalized  infection. 

The  pathogenic  microorganisms  differ  very  much  among  them- 
selves in  the  degree  of  their  disease-inciting  power.  Such  power  is 
known  as  virulence.  Variations  in  virulence  occur,  not  only  among 
different  species  of  pathogenic  bacteria,  but  may  occur  within  the 
same  species.  Pneumococci,  for  instance,  which  have  been  kept  upon 
artificial  media  or  in  other  unfavorable  environment  for  some  time, 
exhibit  less  virulence  than  when  freshly  isolated  from  the  bodies  of 
man  or  animals.  It  is  necessary,  therefore,  in  order  to  produce  infec- 
tion, that  the  particular  bacterium  involved  shall  possess  sufficient 
virulence. 

Whether  or  not  infection  occurs  depends  also  upon  the  number  of 
bacteria  which  gain  entrance  to  the  animal  tissues.  A  small  number 
of  bacteria,  even  though  of  proper  species  and  of  sufficient  virulence, 
may  easily  be  overcome  by  the  first  onslaught  of  the  defensive  forces 
of  the  body.  Bacteria,  therefore,  must  be  in  sufficient  number  to 
overcome  local  defenses  and  to  gain  a  definite  foothold  and  carry 
on  their  life  processes,  before  they  can  give  rise  to  an  infection. 
The  more  virulent  the  germ,  other  conditions  being  equal,  the  smaller 
the  number  necessary  for  the  production  of  disease.  The  introduc- 
tion of  a  single  individual  of  the  anthrax  species,  it  is  claimed,  is 
often  sufficient  to  cause  fatal  infection ;  while  forms  less  well  adapted 
to  the  parasitic  mode  of  life  will  gain  a  foothold  in  the  animal  body 
only  after  the  introduction  of  large  numbers. 

The  Path  of  Infection.— The  portal  by  which  bacteria  gain  en- 
trance to  the  human  body  is  of  great  importance  in  determining 
whether  or  not  disease  shall  occur.  Typhoid  bacilli  rubbed  into 
the  abraded  skin  may  give  rise  to  no  reaction  of  importance,  while 
the  same  microorganism,  if  swallowed,  may  cause  fatal  infection. 
Conversely,  virulent  streptococci,  when  swallowed,  may  cause  no 


FACTORS  OF  PATHOGENICITY  AND   INFECTION  233 

harmful  effects,  while  the  same  bacteria  rubbed  into  the  skin  may 
give  rise  to  a  severe  reaction. 

Animals  and  man  are  protected  against  invasion  by  bacteria  in 
various  ways.  Externally  the  body  is  guarded  by  its  coverings  of 
skin  and  mucous  membranes.  When  these  are  healthy  and  undis- 
turbed, microorganisms  are  usually  held  at  bay.  While  this  is  true 
in  a  general  way  bacteria  may  in  occasional  cases  pass  through 
uninjured  skin  and  mucosa.  Thus  the  Austrian  Plague  Commission 
found  that  guinea-pigs  could  be  infected  when  plague  bacilli  were 
rubbed  into  the  shaven  skin,  and  there  can  hardly  be  much  doubt 
of  the  fact  that  tubercle  bacilli  may  occasionally  pass  through  the 
intestinal  mucosa  into  the  lymphatics  without  causing  local  lesions. 

Even  after  bacteria  of  a  pathogenic  species,  in  large  numbers 
and  of  adequate  virulence,  have  passed  through  a  locally  undefended 
area  in  the  skin  or  mucosa  of  an  animal  or  a  human  being  by  a 
path  most  favorably  adapted  to  them,  it  is  by  no  means  certain 
that  an  infection  will  take  place.  The  bodies  of  animals  and  of 
man  have,  as  we  shall  see,  at  their  disposal  certain  general,  systemic 
weapons  of  defense,  both  in  the  blood  serum  and  the  cellular  ele- 
ments of  blood  and  tissues  which,  if  normally  vigorous  and  active, 
will  usually  overcome  a  certain  number  of  the  invading  bacteria. 
If  these  defenses  are  abnormally  depressed,  or  the  invading  micro- 
organisms are  disproportionately  virulent  or  plentiful,  infection 
takes  place. 

Bacteria,  after  gaining  an  entrance  to  the  body,  may  give  rise 
merely  to  local  inflammation,  necrosis,  and  abscess  formation.  They 
may,  on  the  other  hand,  from  the  local  lesion,  gain  entrance  into  the 
lymphatics  and  blood-vessels  and  be  carried  freely  into  the  circula- 
tion, where,  if  they  survive,  the  resulting  condition  is  known  as 
bacteremia  or  septicemia.  Carried  by  the  blood  to  other  parts  of 
fhe  body,  they  may,  under  favorable  circumstances,  gain  foothold  in 
various  organs  and  give  rise  to  secondary  foci  of  inflammation,  necro- 
sis,  and  abscess  formation.  Such  a  condition  is  known  as  pyemia. 
The  disease  processes  arising  as  the  result  of  bacterial  invasion  may 
depend  wholly  or  in  part  upon  the  mechanical  injury  produced  by 
the  process  of  inflammation,  the  disturbance  of  function  caused  by 
the  presence  of  the  bacteria  in  the  capillaries  and  tissue  spaces,  and 
the  absorption  of  the  necrotic  products  resulting  from  the  reaction 
between  the  body  cells  and  the  microorganisms.  To  a  large  extent, 
however,  infectious  diseases  are  characterized  by  the  symptoms  result- 


234  INFECTION  AND  IMMUNITY 

ing  from  the  absorption  or  diffusion  of  the  poisons  produced  by  the 
bacteria  themselves. 

Bacterial  Poisons. — It  was  plain,  even  to  the  earliest  students 
of  this  subject,  that  mere  mechanical  capillary  obstruction  or  the 
absorption  of  the  products  of  a  local  inflammation  were  insufficient 
to  explain  the  profound  systemic  disturbances  which  accompany 
many  bacterial  infections.  The  very  nature .  of  bacterial  disease, 
therefore,  suggested  the  presence  of  poisons. 

It  was  in  his  investigations  into  the  nature  of  these  poisons  that 
Brieger1  was  led  to  the  discovery  of  the  ptomains.  These  bodies,  first 
isolated  by  him  from  decomposing  beef,  fish,  and  human  cadavers, 
have  found  more  extended  discussion  in  another  section.  Accurately 
classified,  they  are  not  true  bacterial  poisons  in  the  sense  in  which 
the  term  is  now  employed.  Although  it  is  true  that  they  are  produced 
from  protein  material  by  bacterial  action,  they  are  cleavage  products 
derived  from  the  culture  medium  upon  the  composition  of  which 
their  nature  intimately  depends.  The  bacterial  poisons  proper,  on 
the  other  hand,  are  specific  products  of  the  bacteria  themselves, 
dependent  upon  the  nature  of  the  medium  only  as  it  favors  or  retards 
the  full  development  of  the  physiological  functions  of  the  micro- 
organisms. The  poisons,  produced  to  a  greater  or  lesser  extent  by  all 
pathogenic  microorganisms,  may  be  of  several  kinds.  The  true  toxins, 
in  the  specialized  meaning  which  the  term  has  acquired,  are  soluble, 
truly  secretory  products  of  the  bacterial  cells,  passing  from  them 
into  the  culture  medium  during  their  life.  They  may  be  obtained 
free  from  the  bacteria  by  filtration  and  in  a  purer  state  from  the 
filtrates  by  chemical  precipitation  and  a  variety  of  other  methods. 
The  most  important  examples  of  such  poisons  are  those  elaborated 
by  Bacillus  diphtherias  and  Bacillus  tetani.  If  cultures  of  these 
bacteria  or  of  others  of  this  class  are  grown  in  fluid  media  for  several 
days  and  the  medium  is  then  filtered  through  porcelain  candles,  the 
filtrate  will  be  found  toxic  often  to  a  high  degree,  while  the  residue 
will  be  either  inactive  or  comparatively  weak.  Moreover,  if  the  residue 
possesses  any  toxicity  at  all,  the  symptoms  evidencing  this  will  be 
different  from  those  produced  by  the  filtrate. 

There  are  other  microorganisms,  however,  notably  the  cholera 
spirillum  and  the  typhoid  bacillus,  in  which  no  such  exotoxins  are 
formed.  If  these  bacteria  are  cultivated  and  separated  from  the  cul- 


1  Brieger,  "Die  Ptomaine,"  Berlin,  1885  and  1886. 


FACTORS  OF  PATHOGENICITY  AND   INFECTION  235 

ture  fluid  by  filtration,  as  above,  the  fluid  filtrate  will  be  toxic  to 
only  a  very  slight  degree,  whereas  the  residue  may  prove  very  poison- 
ous. In  these  cases,  we  are  dealing,  evidently,  with  poisons  not 
secreted  into  the  medium  by  the  bacteria,  but  rather  attached  more 
or  less  firmly  to  the  bacterial  body.  Such  poisons,  separable  from  the 
bacteria  only  after  death  by  some  method  of  extraction,  or  by  autolysis, 
were  termed  by  Pfeiffer  endotoxins.  The  greater  number  of  the 
pathogenic  bacteria  seem  to  act  chiefly  by  means  of  poisons  of  this 
class.  The  first  to  call  attention  to  the  existence  of  such  intracellular 
poisons  was  Buchner,  who  formulated  his  conclusions  from  the  results 
of  experiments  made  with  a  number  of  microorganisms,  notably  the 
Friedlander  bacillus  and  Staphylococcus  pyogenes  aureus,  with  dead 
cultures  of  which  he  induced  the  formation  of  sterile  abscesses  in 
animals  and  symptoms  of  toxemia.  The  conception  of  "endotoxins," 
received  its  clearest  and  most  definite  expression  in  the  work  of 
Pfeiffer2  on  cholera  poison. 

Some  clarity  of  conception,  based  on  visual  perception,  may  pos- 
sibly be  gained  by  comparing  some  of  the  products  of  pathogenic 
bacteria  with  bacterial  pigments  and  with  insoluble  interstitial  or 
intercellular  substance,  which  may  be  seen  accompanying  bacteria  in 
cover-glass  preparations.  Soluble  toxic  secretions  are  to  be  compared 
to  such  pigments  as  the  pyocyanin  of  Bacillus  pyocyaneus,  which 
is  so  readily  soluble  in  culture  media ;  endotoxins  proper,  to  pigments 
confined  to  the  bacterial  cell,  or  at  least,  when  secreted,  being  insoluble 
in  culture  media,  such  for  instance  as  the  well-known  red  pigment 
of  Bacillus  prodigiosus,  which  may  often  be  seen  free  among  the 
bacteria  in  irregular  red  granules  like  carmine  powder.  That  bodies 
such  as  this  latter  might  be  extruded  from  pathogenic  bacteria  and 
not  be  soluble  in  the  usual  culture  fluids,  is  not  improbable,  and  the 
fact  that  more  or  less  insoluble  interstitial  substances  are  not  infre- 
quent among  bacteria  is  well  known. 

In  all  bacterial  bodies,  after  removal  of  toxins  and  endotoxins,  a 
certain  protein  residue  remains  which,  if  injected  into  animals,  may 
give  rise  to  localized  lesions  such  as  abscesses  or  merely  slight  temporary 
inflammations.  The  nature  of  this  residue  has  been  carefully  studied, 
especially  by  Buchner,  who  has  named  it  bacterial  protein  and  he 
believes  the  substance  to  be  approximately  the  same  in  all  bacteria, 
without  specific  toxic  action,  but  with  a  general  ability  to  exert  a 

2  Pfeiffer,  Zeit.  f.  Hyg.,  xl,  1892. 


236  INFECTION  AND  IMMUNITY 

positive  chemotactic  effect  on  the  white  blood  cells,  thereby  causing 
the  formation  of  pus.  The  nature  of  the  bacterial  proteins  is  by 
no  means  clear,  and  it  is  still  in  doubt  whether  the  separation  of 
these  substances  from  the  endotoxins  can  be  upheld. 

A  number  of  bacteria  may  give  rise  to  both  varieties  of  poisons. 
Thus,  recently,  Kraus  has  claimed  the  discovery  of  a  soluble  toxin 
for  the  cholera  spirillum  and  Doerr  for  the  dysentery  bacillus,  both 
of  which  microorganisms  were  regarded  as  being  purely  of  the 
endotoxin-producing  type. 

It  is  plain,  moreover,  that  occasionally  it  may  be  very  difficult 
to  distinguish  between  a  soluble  toxin  and  an  endotoxin.  In  the 
filtration  experiment  recorded  above,  it  might  well  be  claimed  that 
the  toxicity  of  the  filtrate,  when  not  very  strong,  may  depend  upon 
an  extraction  of  endotoxins  from  the  bodies  of  the  bacteria  by  the 
medium.  The  final  test,  in  such  instances,  lies  in  the  power  of 
true  toxins  to  stimulate  in  animals  the  production  of  antitoxins; 
for,  as  we  shall  see  later,  the  injection  of  true  soluble  toxins  into 
animals  gives  rise  to  antitoxins,  whereas  the  formation  of  such 
neutralizing  bodies  in  the  serum  or  plasma  does  not,  it  is  claimed, 
follow  the  injection  of  endotoxins. 

We  could  spend  much  time  in  analyzing  the  literature  on  the 
exotoxin  and  endotoxin,  and  this,  of  course,  would  be  important 
were  we  attempting  in  this  book  to  cover  completely  immunological 
problems.  When  all  is  said  and  done,  however,  the  present  status 
of  the  question  is  as  follows:  Certain  bacteria,  like  the  diphtheria 
bacillus,  the  tetanus  bacillus,  B.  botulinus,  some  of  the  anaerobes 
of  surgical  infections,  etc.,  produce  secretory  products  during  life 
which  are  highly  toxic,  can  be  obtained  during  the  life  of  the 
culture  by  simple  filtration,  and  which  incite,  in  carefully  treated 
animals,  specific  neutralizing  substances,  or  antitoxins,  which  neu- 
tralize the  action  of  the  toxin,  roughly  according  to  the  law  of  mul- 
tiples. These  antitoxins  in  the  serum,  therefore,  can  be  shown 
definitely  to  prevent  the  injury  of  the  animal  by  the  toxin. 

In  many  bacteria  such  soluble  toxins  cannot  be  demonstrated. 
Older  bacterial  culture  filtrates  and  the  bodies  of  the  bacteria  may 
be  highly  toxic,  as  in  the  case  of  typhoid,  cholera,  and  practically 
all  the  Gram-negative  organisms,  but  these  toxic  substances  are 
derived  either  from  direct  extraction  of  the  bacterial  bodies,  or,  as 
claimed  by  some,  may  represent  split  products  produced  in  the 
course  of  decomposition  or  cleavage  of  the  bacterial  protein.  These 


FACTORS  OF  PATHOGENIOITY  AND   INFECTION  2,37 

substances,  called  by  Pfeiffer  endotoxins,  do  not  produce  antitoxins. 
It  is  doubtful,  in  our  minds,  whether  they  may  be  regarded  as 
strictly  specific  in  all  cases. 

In  addition  to  these  poisonous  substances,  the  writer,  with  Kutt- 
ner  and  Parker,3  has  recently  obtained  non-specific  toxic  substances 
for  a  great  many  different  bacteria  (streptococci,  typhoid  bacilli, 
influenza  bacilli,  etc.)  which  appeared  in  cultures  as  early  as  6  to  18 
hours,  could  be  obtained  by  filtration,  and  could  also  be  obtained 
by  washing  young  cultures  on  solid  media  with  salt  solution  and 
filtering.  These  substances  are  not  unlikely  similar  to  those  en- 
countered by  some  other  writers  who  have  interpreted  them  as  true 
exotoxins,  but,  as  far  as  we  can  determine,  they  are  neither  specific 
nor  antigenic.  Nevertheless,  they  are  regular  in  appearance,  suffi- 
ciently potent  to  make  rabbits  very  sick,  though  rarely  to  kill  them, 
and  must  be  taken  into  account  in  all  work  in  which  the  toxic 
substances  of  bacteria  are  studied  by  the  usual  methods.  We  can 
speak  of  them,  for  want  of  more  accurate  definition,  as  bacterial 
"X"  substances. 

In  resistance  to  chemical  action  and  heat,  the  various  poisons 
show  widely  divergent  properties.  As  a  general  rule,  most  true 
soluble  toxins  are  delicately  thermolabile,  they  are  destroyed  by 
moderate  heating,  and  deteriorate  easily  on  standing.  Their  chemical 
nature  is  by  no  means  clear,  but,  on  precipitation  of  toxic  solutions 
with  magnesium  sulphate,  these  poisons  come  down  together  with 
the  globulins.  The  nature  of  the  "endotoxins"  is  still  less  clearly 
understood.  Most  of  them  are  far  less  liabile  than  the  extracellular 
poisons.  Some  powerful  intracellular  poisons,  like  those  of  the 
Gartner  bacillus  of  meat  poisoning  and  the  poison  attached  to  the 
bodies  of  typhoid  bacilli  may  undergo  exposure  to  even  100°  C.  and 
still  retain  their  toxic  properties.  The  nature  of  each  individual 
poison  will  be  discussed  in  connection  with  its  microorganism. 

It  should  be  remembered,  moreover,  by  those  studying  bacterial 
poisons  that  recent  investigations  have  shown  that  a  number  of 
bacteria  (coli,  influenza,  etc.)  may  produce  substances  either  identical 
with  or  closely  related  to  histamin  and  tyramin,  on  pepton  media, 
after  five  or  more  days  of  growth. 


97insftcr,  Parker  and  Kuttner,  Transact.  Soc.  for  Exp.  Biol.  and  Mod.,  Jan., 
1921. 


238  INFECTION   AND   IMMUNITY 

The  Made  of  Action  of  Bacterial  Poisons. — Close  study  of  the 
toxic  products  of  various  microorganisms  has  shown  that  many  of 
the  bacterial  poisons  possess  a  more  or  less  definite  selective  action 
upon  special  tissues  and  organs.  Thus,  certain  soluble  toxins  of  the 
tetanus  bacillus  and  Bacillus  botulinus  attack  specifically  the  nervous 
system.  Again,  certain  poisons  elaborated  by  the  staphylococci,  the 
tetanus  bacillus,  the  streptococci,  and  other  germs,  the  so-called 
1 '  hemolysins, ' '  attack  primarily  the  red  blood  corpuscles.  Other 
poisons  again  act  on  the  white  blood  corpuscles ;  in  short,  the  char- 
acteristic affinity  of  specific  bacterial  poisons  for  certain  organs  is 
a  widely  recognized  fact. 

In  explanation  of  this  behavior,  much  aid  has  been  given  by  the 
researches  of  Meyer,4  Overton,5  Ehrlich,6  and  others  upon  the  causes 
for  the  analogous  selective  behavior  of  various  narcotics  and  alka- 
loids. It  seems  probable,  from  the  researches  of  these  men,  that 
the  selective  action  of  poisons  depends  upon  the  ability,  chemical 
or  physical  or  both,  of  the  poisons  to  enter  into  combination  with 
the  specifically  affected  cells.  From  the  nature  of  the  combinations 
formed,  it  seems  not  unlikely  that  the  physical  factors,  such  as 
solubility  in  the  cell  plasma,  may  also  play  an  important  part. 

Observations  of  a  more  purely  bacteriological  nature  have  tended 
to  bear  out  these  conclusions.  Wassermann  and  Takaki,7  for  in- 
stance, have  shown  that  tetanus  toxin,  which  specifically  attacks 
the  nervous  system,  may  be  removed  from  solution  by  the  addition 
of  brain  substance.  Removal  of  the  brain  tissue  by  centrifugation 
leaves  the  solution  free  from  toxin.  In  the  same  way  it  has  been 
shown  that  hemolytic  poisons  can  be  removed  from  solutions  by 
contact  with  red  blood  cells,  but  only  when  the  red  blood  cells 
of  susceptible  species  are  employed. 

Similar  observations  have  been  made  in  the  case  of  leukocidin, 
a  bacterial  poison  acting  upon  the  white  blood  cells  specifically.8 

That  bacterial  poisons  injected  into  susceptible  animals  rapidly 
disappear  from  the  circulation  is  a  fact  which  bears  out  the  view 
that  a  combination  between  affected  tissue  and  toxin  must  take 


*  Meyer,  Arch.  f.  exper.  Pathol.,  1899,  1901. 

*  Overton,  "Studien  iib.  d.  Narkose,"  Jena,  1901. 

9  Ehrlich,  ' '  Sauerstoffs-Bediirfniss  des  Organismus,"  Berlin,  1885. 
7  Wassermann  und  Takaki,  Berl.  klin.  Woch.,   1898. 
9  Sachs,  Hof meister 's  Beitrage,  11,  1902.  ' 


FACTORS  OF  PATHOGENICITY  AND  INFECTION  239 

place.  Db'nitz,9  for  instance,  has  shown  that  within  four  to  eight 
minutes  after  the  injection  of  certain  toxins,  considerable  quantities 
will  have  disappeared  from  the  circulation.  Conversely,  Metchni- 
koff10  has  observed  that  tetanus  toxin  injected  into  insusceptible 
animals  (lizards)  may  be  detected  in  the  blood  stream  for  as  long 
as  two  months  after  administration. 


9  Ddnitz,  Deut.  mod.  Woch.,  1897. 

10Metchnikoff,  "L'immunite  dans  les  malad.  infect." 


CHAPTER  XII 

DEFENSIVE  FACTOKS  OF  THE  ANIMAL  ORGANISM 

GENERAL    CONSIDERATIONS 

WE  have  seen  that  the  mere  entrance  of  a  pathogenic  microor- 
ganism into  the  human  or  animal  body  through  a  breach  in  the  con- 
tinuity of  the  mechanical  defenses  of  skin  or  mucosa  does  not  neces- 
sarily lead  to  the  development  of  an  infection.  The  opportunities  for 
such  an  invasion  are  so  numerous,  and  the  contact  of  members  of 
the  animal  kingdom  with  the  germs  of  disease  is  so  constant,  that  if 
this  were  the  case,  sooner  or  later  all  would  succumb.  It  is  plain, 
therefore,  that  the  animal  body  must  possess  more  subtle  means  of 
defense,  by  virtue  of  which  pathogenic  germs  are,  even  after  their 
entrance  into  the  tissues  and  fluids,  disposed  of,  or  at  least  prevented 
from  proliferating  and  elaborating  their  poisons.  The  power  which 
enables  the  body  to  accomplish  this  is  spoken  of  as  resistance.  When 
this  resistance,  which  in  some  degree  is  common  to  all  members  of 
the  animal  kingdom,  is  especially  marked,  it  is  spoken  of  as  "  im- 
munity. '  ' 

From  this  it  follows  naturally  that  the  terms  resistance  and  im- 
munity, as  well  as  their  converse,  susceptibility,  are  relative  and  not 
absolute  terms.  Degrees  of  resistance  exist,  which  are  determined 
to  a  certain  extent  by  individual,  racial,  or  species  peculiarities ;  and 
persons  or  animals  are  spoken  of  as  immune  when  they  are  unaffected 
by  an  exposure  or  an  inoculation  to  which  the  normal  average  in- 
dividual of  the  same  species  would  ordinarily  succumb.  The  word 
does  not  imply,  however,  that  these  individuals  could  not  be  infected 
with  unusually  virulent  or  large  doses,  or  under  particularly  unfavor- 
able circumstances.  Thus,  birds,  while  immune  against  the  ordinary 
dangers  of  tetanus  bacilli,  may  be  killed  by  experimental  inoculations 
with  very  large  doses  of  tetanus  toxin.1  Similarly,  Pasteur  rendered 


'Quoted  from  Abel,  Kolle  und  Wassermann,  "Handbuch,"  etc. 

240 


DEFENSIVE   FACTORS  OF  THE   ANIMAL  ORGANISM          241 

naturally  immune  hens  susceptible  to  anthrax  by  cooling  them  to  a 
subnormal  temperature,  and  Canalis  and  Morpurgo  did  the  same  with 
doves  by  subjecting  them  to  starvation. 

Absolute  immunity  is  exceedingly  rare.  The  entire  insusceptibility 
of  cold-blooded  animals  (frogs  and  turtles)  under  normal  conditions 
to  inoculation  with  even  the  largest  doses  of  many  of  the  bacteria 
pathogenic  for  warm-blooded  animals,  and  the  immunity  of  all  the 
lower  animals  against  leprosy,  are  among  the  few  instances  of  absolute 
immunity  known.2  Apart  from  such  exceptional  cases,  however,  re- 
sistance, immunity,  and  susceptibility  must  be  regarded  as  purely 
relative  terms. 

The  power  of  resisting  any  specific  infection  may  be  the  natural 
heritage  of  a  race  or  species,  and  is  then  spoken  of  as  natural  im- 
munity. It  may,  on  the  other  hand,  he  acquired  either  accidentally 
or  artificially  by  a  member  of  an  ordinarily  susceptible  species,  and 
is  then  called  acquired  immunity. 

Natural  Immunity. — SPECIES  IMMUNITY. — It  is  well  known  that 
many  of  the  infectious  diseases  which  commonly  affect  man,  do  not, 
so  far  as  we  know,  occur  spontaneously  in  animals.  Thus,  infection 
with  B.  typhosus,  the  vibrio  of  cholera,  or  the  meningococcus  occurs 
in  animals  only  after  experimental  inoculation.  Gonorrheal  and 
syphilitic  infection,  furthermore,  not  only  does  not  occur  spontane- 
ously, but  is  produced  experimentally  in  animals  with  the  greatest 
difficulty — the  consequent  diseases  being  incomparably  milder  than 
those  occurring  in  man.  Other  diseases,  like  leprosy,  influenza,  and 
the  exanthemata,3  have  never  been  sucessfully  transmitted  to  animals. 

Conversely,  there  are  diseases  among  animals  which  do  not  spon- 
taneously attack  man.  Thus,  human  beings  enjoy  immunity  against 
Rinderpest,  and,  to  a  lesser  degree,  against  chicken  cholera. 

Among  animal  species  themselves  great  differences  in  susceptibility 
and  resistance  toward  the  various  infections  exist.  Often-quoted 
examples  of  this  are  the  remarkable  resistance  to  anthrax  of  rats  and 
dogs,  and  the  immunity  of  the  common  fowl  against  tetanus. 

The  factors  which  determine  these  differences  of  susceptibility  and 
resistance  among  the  various  species  are  not  clearly  understood.  It 
has  been  suggested  that  diet  in  some  instances  may  influence  these 
relations,  inasmuch  as  carnivorous  animals  are  often  highly  resistant 

2  Lubarsch,  Zeit.  f.  klin.  Medi/.,  xix. 
'With  the  possible  exception  of  smallpox. 


242  INFECTION  AND  IMMUNITY 

to  glanders,  anthrax,  and  even  tuberculous  infections,  to  which  herbiv- 
orous animals  are  markedly  susceptible.4  It  is  likely,  too,  that  the 
great  differences  between  animals  of  various  species  in  their  metab- 
olism, temperature,  etc.,  may  call  for  special  cultural  adaptation  on 
the  part  of  the  bacteria.  The  fact  that  the  bacillus  of  avian  tuber- 
culosis— whose  natural  host  has  a  normal  body  temperature  of  40°  C. 
and  above — will  grow  on  culture  media  at  40  to  50°  C.,  whereas  B. 
tuberculosis  of  man  can  not  be  cultivated  at  a  temperature  above 
40°  C.,  would  seem  to  lend  some  support  to  this  view.  The  difference 
between  warm-  and  cold-blooded  animals  has  already  been  noted.  The 
necessity  for  cultural  adaptation,  too,  would  seem  to  be  borne  out 
by  the  great  enhancement  observed  in  the  virulence  of  certain  micro- 
organisms for  a  given  species  after  repeated  passage  through  in- 
dividuals of  this  species. 

RACIAL  IMMUNITY. — Just  as  differences  in  susceptibility  and  im- 
munity exist  among  the  various  animal  species,  so  the  separate  races 
or  varieties  within  the  same  species  may  display  differences  in  their 
reactions  toward  pathogenic  germs.  Algerian  sheep,  for  instance, 
show  a  much  higher  resistance  to  anthrax  than  do  our  own  domestic 
sheep,  and  the  various  races  of  mice  differ  in  their  susceptibility  to 
anthrax  and  to  glanders. 

Similar  racial  differences  are  common  among  human  beings.  As 
a  general  rule,  it  may  be  said  that  a  race  among  whom  a  certain 
disease  has  been  endemic  for  many  ages  is  less  susceptible  to  this  dis- 
ease than  are  other  races  among  whom  it  has  been  more  recently 
introduced.  The  appalling  ravages  of  tuberculosis  among  negroes, 
American  Indians,  and  Esquimaux,  bear  striking  witness  to  this  fact. 
Conversely,  the  comparative  immunity  of  the  negro  from  yellow  fever, 
a  disease  of  the  greatest  virulence  for  Caucasians,  furnishes  further 
evidence  in  favor  of  this  opinion.  It  must  not  be  forgotten,  however, 
in  judging  of  these  relations,  that  the  great  differences  in  the  customs 
of  personal  and  social  hygiene  existing  among  the  various  races  may 
considerably  affect  the  transmission  of  disease  and  lead  to  false  con- 
clusions. 

In  so  far  as  the  statement  made  above  is  true,  however,  it  seems 
to  indicate  that  the  endemic  diseases  have  carried  in  their  train  a 
certain  .degree  of  inherited  immunity. 


'  Hahn,  in  Kolle  und  Wassermann,  vol.  iv. 


DEFENSIVE   FACTORS  OF  THE  ANIMAL  ORGANISM          243 

In  other  cases5 — as  in.  the  instance  of  the  malaria-immunity  of 
negroes — the  resistance  seems  to  be  acquired  rather  than  inherited, 
for,  as  Hirsch  was  first  to  note,  death  from  this  disease  occurred 
frequently  among  the  children,  while  adult  negroes  were  rarely 
attacked. 

DIFFERENCES  IN  INDIVIDUAL  RESISTANCE. — In  bacteriological  ex- 
perimentation with  smaller  test  animals,  a  direct  ratio  may  often 
exist  between  body  weight  and  dosage  in  determining  the  outcome  of 
an  infection,  provided  the  mode  of  inoculation  has  been  the  same 
and  the  virulence  of  the  germ  not  excessive.  It  would  seem,  therefore, 
that  among  these  animals  the  difference  in  resistance  in  the  face  of 
an  artificial  infection  between  individuals  of  the  same  race  is  very 
slight. 

In  higher  animals,  however,  especially  in  the  case  of  man,  the 
existence  of  such  apparent  individual  differences  is  a  well-established 
fact,  although  in  judging  of  them  we  must  not  forget  that  the  condi- 
tions of  infection  are  not  subject  to  the  uniformity  and  control  which 
animal  experimentation  permits.  Of  a  number  of  persons  exposed  to 
any  given  infection  there  are  always  some  who  are  entirely  unaffected 
and  there  are  great  variations  in  the  severity  of  the  disease  in  those 
who  are  attacked.  In  the  absence  of  positive  evidence  in  support  of 
the  direct  inheritance  of  this  individual  immunity,  the  most  reasonable 
explanation  for  such  differences  in  resistance  seems  to  lie  in  attributing 
them  to  individual  variations  in  metabolism  or  body  chemistry.  De- 
pressions, for  instance,  in  the  acidity  of  the  gastric  secretion  would 
predispose  to  certain  infections  of  gastro-intestinal  origin.  Anatomical 
differences,  too,  may  possibly  influence  resistance.  Thus,  Birch-Hirsch- 
feld  believed  that  certain  anomalous  arrangements  of  the  bronchial 
tubes  predisposed  to  tuberculosis. 

Instances  of  transient  susceptibility  induced  by  physical  or  mental 
overwork,  starvation,  etc.,  should  hardly  be  classified  under  this  head- 
ing, since  the  conditions  in  such  cases  correspond  simply  to  experi- 
mental depression  of  natural  species  for  race  resistance. 

Acquired  Immunity. — It  is  a  matter  of  common  experience  that 
many  of  the  infectious  diseases  occur  but  once  in  the  same  individual. 
This  is  notably  the  case  with  typhoid  fever,  yellow  fever,  and  most 
of  the  exanthemata,  and  is  too  general  an  observation  to  require  exten- 
sive illustration.  A  single  attack  of  any  of  the  diseases  of  this  class 

6  Halin,  in  Kolle  und  Wassermann,  loc.  cit. 


244  INFECTION   AND   IMMUNITY 

alters  in  some  way  the  resistance  of  the  individual  so  that  further 
exposure  to  the  infective  agent  is  usually  without  menace,  either  for 
a  limited  period  after  the  attack,  or  for  life.  Resistance  acquired  in 
this  way  is  often  spoken  of  as  acquired  immunity. 

The  protection  conferred  by  certain  diseases  against  further  attack 
was  recognized  many  centuries  ago,  and  there  are  records  which  show 
that  attempts  were  made  in  ancient  China  and  India  to  inoculate 
healthy  individuals  with  pus  from  small-pox  pustules  in  the  hope 
of  producing  by  this  process  a  mild  form  of  the  disease  and  its 
consequent  immunity. 

Pasteur,  before  all  others,  thought  philosophically  about  the 
phenomena  of  acquired  immunity,  and,  with  adequate  knowledge, 
realized  the  possibility  of  artificially  bestowing  immunity  without 
inflicting  the  dangers  of  the  fully  potent  infective  agent.  The  first 
observation  which,  made  by  him,  purely  accidentally,  inspired  the 
hope  of  the  achievement  of  such  a  result,  occurred  during  his  experi- 
ments with  chicken  cholera.  The  failure  of  animals  to  die  after  inocula- 
tion with  an  old  culture  of  the  bacilli  of  chicken  cholera,  fully  potent 
but  a  few  weeks  previously,  pointed  to  the  attenuation  of  these  bacilli 
by  their  prolonged  cultivation  without  transplantation.  With  this 
observation  as  a  point  of  departure  he  carried  out  a  series  of  investiga- 
tions with  the  purpose  of  discovering  a  method  of  so  weakening  or 
attenuating  various  incitants  of  disease  that  they  could  be  introduced 
into  susceptible  individuals  without  endangering  life  and  yet  without 
losing  their  property  of  conferring  protection.  The  brilliant  results 
achieved  by  Jenner,  many  years  before,  in  protecting  against  smallpox 
by  inoculating  with  the  entirely  innocuous  products  of  the  pustules 
of  cowpox  furnished  an  analogy  which  gave  much  encouraging  support 
to  this  prospect. 

The  experimental  work  which  Pasteur  carried  out  to  solve  this 
problem  not  only  reaped  a  rich  harvest  of  facts,  but  gave  to  science 
the  first  and  brilliant  examples  of  the  application  of  exact  laboratory 
methods  to  problems  of  immunity. 

ACTIVE    IMMUNITY 

Active  Artificial  Immunity. — The  process  of  conferring  protec- 
tion by  treatment  with  either  an  attenuated  form  or  a  sublethal  quan- 
tity of  the  infectious  agent  of  a  disease,  or  its  products,  is  spoken  of 


DEFENSIVE   FACTORS  OF  THE  ANIMAL  ORGANISM          245 

Whatever  the  method  employed,  the  immunized  individuals  gain 
their  power  of  resistance  by  the  unaided  reactions  of  their  own  tissues. 
They  themselves  take  an  active  physiological  part  in  the  acquisition 
of  this  new  property  of  immunity.  For  this  reason,  Ehrlich  has  aptly 
termed  these  processes  * '  active  immunization. ' ' 

There  are  various  methods  by  which  this  can  be  accomplished,  all 
of  which  were,  in  actual  application  or  in  principle,  discovered  by 
Pasteur  and  his  associates,  and  can  be  best  reviewed  by  a  study  of 
their  work. 

ACTIVE  IMMUNIZATION  WITH  ATTENUATED  CULTURES. — In  the 
course  of  his  experiments  upon  chicken  cholera,  as  mentioned  above, 
Pasteur6  accidentally  discovered  that  the  virulence  of  the  bacilli  of 
this  disease  was  greatly  reduced  by  prolonged  cultivation  upon  artifi- 
cial -media.  This  was  especially  noticeable  in  broth  cultures  which 
had  been  stored  for  long  periods  without  transplantation.  By  repeated 
injections  of  such  cultures  into  fowl,  he  succeeded  in  rendering  the 
animals  immune  against  subsequent  inoculations  with  lethal  doses  of 
fully  virulent  strains. 

During  the  same  year,  1880,  in  which  Pasteur  published  his  ob- 
servations on  chicken  cholera,  Toussaint7  succeeded  in  immunizing 
sheep  against  anthrax  by  inoculating  them  with  blood  from  infected 
animals,  defibrinated  and  heated  to  55°  C.  for  ten  minutes.  Toussaint 
wrongly  believed,  however,  that  the  blood  which  had  been  used  in 
his  immunizations  was  free  from  living  bacteria.  In  repeating  this 
work  Pasteur  showed  that  the  protection  in  Toussaint 's  cases  was 
conferred  by  living  bacteria,  the  virulence  of  which  had  been  reduced 
by  their  subjection  to  heat. 

In  following  out  the  suggestions  offered  by  these  experiments, 
Pasteur8  discovered  that  he  could  reduce  the  virulence  of  anthrax 
bacilli  much  more  reliably  than  by  Toussaint 's  method,  by  cultivating 
the  organisms  at  increased  temperatures  (42°  to  43°  C.).  By  this 
process  of  attenuation  he  was  able  to  produce  "vaccines"  of  roughly 
measurable  strength,  with  which  he  succeeded  in  immunizing  sheep 
and  cattle.  A  successful  demonstration  of  his  discovery  was  made 
by  him  at  Pouilly-le-Fort,  soon  after,  upon  a  large  number  of  animals 
and  before  a  commission  of  professional  men. 


6  Pasteur,  Compt.  rend,  do  1'acad.  des  sci.,  1880,  t.  xc. 

7  Toussaint,  Compt.  rend,  de  1'acad.  des  sci.,  1880,  t.  xei. 

*  Pasteur,  Chamberland  et  Eoux,  Compt.  rend,  de  1'acad.  des  sci.,  1881,  t.  xcii. 


246  INFECTION  A^D   IMMUNITY 

It  is  a  fact  well  known  to  bacteriologists  that  certain  of  the  patho- 
genic microorganisms,  when  passed  through  several  individuals  of 
the  same  animal  species,  become  gradually  more  virulent  for  this 
species.  In  his  studies  on  the  bacillus  of  hog  cholera,  Pasteur9  ob- 
served that  when  this  microorganism  was  passed  through  the  bodies 
of  several  rabbits  it  gained  in  virulence  for  rabbits,  but  became  less 
potent  against  hogs.  He  succeeded,  subsequently,  in  protecting  hogs 
against  fully  virulent  cultures  by  treating  them  with  strains  which 
had  been  attenuated  by  their  passage  through  rabbits. 

A  further  principle  of  attenuation  for  purposes  of  immunization 
was,  at  about  this  time,  contributed  by  Chamberland  and  Roux,10 
who  reduced  the  virulence  of  anthrax  cultures  by  growing  them  in 
the  presence  of  weak  antiseptics  (carbolic  acid  1:600,  potassium 
bichromate  1:5,000,  or  sulphuric  acid  1:200).  Cultivated  under 
such  conditions  the  bacilli  lost  their  ability  to  form  spores  and  became 
entirely  avirulent  for  sheep  within  ten  days.  A  similar  result  was 
later  obtained  by  Behring11  when  attenuating  B.  diphtherias  cultures 
by  the  addition  of  terchlorid  of  iodin. 

ACTIVE  IMMUNIZATION  WITH  SUBLETHAL  DOSES  OF  FULLY  VIRULENT 
BACTERIA. — The  use  of  fully  virulent  microorganisms  in  minute  quan- 
tities for  purposes  of  immunization  was  first  suggested  by  Chauveau,12 
and  is  naturally  inapplicable  to  extremely  virulent  organisms  like  B. 
anthracis.  The  principle,  however,  is  perfectly  valid,  and  has  been 
experimentally  applied  by  many  observers,  notably  by  Ferran13  in 
the  case  of  cholera.  A  similar  method  proved  of  practical  value  in 
the  hands  of  Theobald  Smith  and  Kilborne14  in  prophylaxis  against 
the  protozoan  disease,  Texas  fever. 

ACTIVE  IMMUNIZATION  WITH  DEAD  BACTERIA. — Suggested  by  Chau- 
veau, the  method  of  active  immunization  with  gradually  increasing 
doses  of  dead  microorganisms  has  been  successfully  employed  by 
various  observers,  chief  among  whom  are  Pfeiffer,  Brieger,  Wright, 
and  Wassermann.  The  method  is  especially  useful  against  that  class 
of  bacteria  in  which  the  cell  bodies  (endotoxins)  have  been  found  to 


9  Pasteur,  Compt.  rend,  de  1'acad.  des  sci.,  1882,  t.  xcv. 

10  CJiamberland  et  Eoux,  Compt.  rend,  de  1'acad.  dcs  sci.,  1882,  t.  xcvi. 
u  Behring,  Zcit.   f.  Hyg.,  xii,  1892. 

™Chauveau,  Compt.  rend,  de  Pacad.  des  sci.,  1881,  t.  xcii. 
"Ferran,  Compt.  rend,  de  1'acad.  des  sci.,  1895,  t.  ci. 

14  Th.  Smith  and  Kilborne,  U.  S.  Dept.  of  Agri.,  Bureau  of  Ani.  Indust.,  Wash., 
1893. 


DEFENSIVE   FACTORS  OF  THE  ANIMAL  ORGANISM          247 

be  incomparably  more  poisonous  than  their  extracellular  products 
(toxins) .  From  a  practical  point  of  view,  the  method  is  of  the  greatest 
importance  in  routine  laboratory  immunization  against  B.  typhosus, 
Vibrio  cholera?  asiaticse,  B.  pestis,  and  a  number  of  other  bacteria. 
In  the  therapy  of  human  disease,  this  method  has  recently  come  into 
great  prominence,  chiefly  through  the  work  of  Wright,  whose  inves- 
tigations will  be  more  fully  discussed  in  a  subsequent  section. 

ACTIVE  IMMUNIZATION  WITH  BACTERIAL  PRODUCTS. — Many  bacteria 
when  grown  in  fluid  media  produce  extracellular,  soluble  poisons  which 
remain  in  the  medium  after  the  microorganisms  have  been  removed 
by  filtration  or  centrifugalization.  Since  the  diseases  caused  by  such 
microorganisms  are,  to  a  large  extent,  due  to  the  soluble  poisons 
excreted  by  them,  animals  can  be  actively  immunized  against  this 
class  of  bacteria  by  the  inoculation  of  gradually  increasing  doses  of 
the  specific  poison  or  toxin.  This  method  is  naturally  most  successful 
against  those  microorganisms  which  possess  the  power  of  toxin  forma- 
tion to  a  highly  developed  degree.  Most  important  among  these  are 
B.  diphtherias  and  B.  tetani.  The  first  successful  application  of  this 
principle  of  active  immunization,  however,  was  made  by  Salmon  and 
Smith15  in  the  case  of  hog  cholera. 


PASSIVE    IMMUNITY 

In  Pasteur's  basic  experiments,  as  in  those  of  the  other  scientists 
who  followed  in  his  footsteps,  the  methods  of  immunization  were  based 
upon  the  development  of  a  high  resistance  in  the  treated  subject  by 
virtue  of  its  own  physiological  activities.  This  process  we  have  spoken 
of  as  "active  immunization"  and  it  is  self-evident  that  a  method  of 
this  kind  can,  in  the  treatment  of  disease,  be  employed  prophylactically 
only  against  possible  infection,  or  in  localized  acute  infections,  or  at 
the  beginning  of  a  long  period  of  incubation  before  actual  symptoms 
have  appeared,  as  in  rabies  or  in  chronic  conditions  in  which  the 
infection  is  not  of  a  severe  or  acute  nature. 

A  new  and  therapeutically  more  hopeful  direction  was  given  to 
the  study  of  immunity  when,  in  1890  and  1892,  v.  Behring  and  his 
collaborators  discovered  that  the  sera  of  animals  immunized  against 


15  Salmon  and  Smith,  Eep.  of  Com.  of  Agri.,  Wash.,  1885  and  1886. 


248  INFECTION   AND  IMMUNITY 

the  toxins  of  tetanus16  and  of  diphtheria17  bacilli  would  protect  normal 
animals  against  the  harmful  action  of  these  poisons.  The  animals 
thus  protected  obviously  had  taken  no  active  part  in  their  own  defense, 
but  were  protected  from  the  action  of  the  poison  by  the  substances 
transferred  to  them  in  the  sera  of  the  actively  immunized  animals. 
Such  immunity  or  protection,  therefore,  is  a  purely  passive  phenom- 
enon so  far  as  the  treated  animal  is  concerned,  and  the  process  is 
for  this  reason  spoken  of  as  "passive  immunization." 

Passive  immunization  of  this  description  is  practically  applicable 
chiefly  against  diseases  caused  by  bacteria  which  produce  powerful 
toxins,  and  the  sera  of  animals  actively  immunized  against  such  toxins 
are  called  antitoxic  sera.  In  the  treatment  of  the  two  diseases  men- 
tioned above,  diphtheria  and  tetanus,  the  respective  antitoxic  sera 
have  reached  broad  and  beneficial  therapeutic  application,  and  in- 
numerable lives  have  been  saved  by  their  use. 

Passive  immunization  against  microorganisms  not  characterized 
by  marked  toxin  formation  was  attempted,  even  before  Behring's 
discovery,  by  Richet  and  Hericourt,18  experimenting  with  cocci,  and 
by  Babes,19  in  the  case  of  rabies ;  and  the  underlying  thought  had 
been  the  basis  of  Toussaint's  work  upon  anthrax.  Microorganisms, 
however,  which  exert  their  harmful  action  rather  by  the  contents 
of  the  bacterial  cells  than  by  secreted,  soluble  toxins,  do  not,  so  far 
as  is  known,  produce  antitoxins  in  the  sera  of  immunized  animals. 
The  substances  which  they  call  forth  in  the  process  are  directed  against 
the  invading  organisms  themselves  in  that  they  possess  the  power 
of  destroying  or  of  causing  dissolution  of  the  specific  germs  used  in 
their  production. 

Such  antibacterial  sera  are  extensively  used  in  the  laboratory  in 
the  passive  immunization  of  animals  against  a  large  number  of  germs, 
and  are  fairly  effectual  when  used  before,  at  the  same  time  with, 
or  soon  after,  infection.  Their  therapeutic  use  in  human  disease, 
however,  has,  up  to  the  present  time,  been  disappointing  and  their 
prophylactic  and  curative  action  has  been  almost  invariably  ineffectual 
or  feeble  at  best,  except  when  the  antibacterial  sera  could  be  brought 
in  direct  contact  with  the  germs,  in  closed  cavities  or  localized  lesions. 

16  v.  Bearing  and  Kitasato,  Dent.  med.  Woeh.,  49,  1890. 

17  v.  Bearing  and  Wernicke,  Zeit.  f .  Hyg.,  1892. 

18  Richet  et  Hericourt,  Compt.  rend,  de  Paead.  dos  sei.,  1888. 

19  Babes  et  Lepp,  Ann.  de  1'inst.  Pasteur,  1889. 


DEFENSIVE   FACTORS  OF  THE  ANIMAL  ORGANISM          249 

Thus,  in  epidemic  meningitis,  such  sera  have  proved  extremely  useful 
in  the  hands  of  Flexner,  when  injected  directly  into  the  spinal  canal. 


ANTIBODIES  AND  THE  SUBSTANCES  GIVING  RISE  TO  THEM 

In  the  foregoing  sections  we  have  seen  that  the  process  of  active 
immunization  so  changes  the  animal  body  that  it  becomes  highly 
resistant  against  an  infection  to  which  it  had  formerly  in  many  in- 
stances been  delicately  susceptible.  In  the  absence  of  visible  anatomical 
or  histological  changes  accompanying  the  acquisition  of  this  new  power, 
investigators,  in  order  to  account  for  it,  were  led  to  examine  the 
physiological  properties  of  the  body  cells  and  fluids  of  immunized 
subjects.  While  it  was  reasonable  to  suppose  that  all  the  cells  and 
tissues  were  affected  by,  or  might  have  taken  part  in,  a  physiological 
change  so  profoundly  influencing  the  individual,  the  blood,  because 
of  its  unquestionably  close  relation  to  inflammatory  reactions,  and 
because  of  the  ease  with  which  it  could  be  obtained  and  studied, 
claimed  the  first  and  closest  attention.  The  bactericidal  properties 
or  normal  blood  serum  noted  in  1886  by  Nuttall,20  v.  Fodor,21  and 
Fliigge,  moreover,  aided  in  pointing  to  this  tissue  as  primarily  the 
seat  of  the  immunizing  agents.  It  is  an  interesting  historical  fact, 
that,  long  before  this  time,  the  English  physician  Hunter  had  noted 
that  blood  did  not  decompose  so  rapidly  as  other  animal  tissues. 

The  study  of  the  blood  serum  of  immunized  animals  as  to  simple 
changes  in  chemical  composition  or  physical  properties  has  shed  little 
light  upon  the  subject.  Beljaeff22  in  a  recent  investigation  found  little 
or  no  alteration  from  the  normal  in  the  blood  sera  of  immunized 
animals  as  to  index  of  refraction,  specific  gravity,  and  alkalinity. 
Joachim23  and  Moll  agree  in  stating  that  immune  blood  serum  is  com- 
paratively richer  in  globulin  than  normal  serum.  Similar  observations 
had  been  made  by  Hiss  and  Atkinson24  and  others.  Important  and 
significant  as  these  purely  chemical  observations  are,  they  have  helped 
little  in  explaining  the  nature  of  the  processes  going  on  in  immune 
sera.  The  first  actual  light  was  thrown  upon  the  mysterious  phenomena 

20  Nuttall,  Zeit.  f.  Hyg.,  i,  1886. 

21  v.  Fodor,  Deut.  med.  Woch.,  1886. 
"Beljaeff,  Cent.  f.  Bakt.,  xxxiii. 

23  Joachim,  Pfliigers  Archiv,  xciii. 

-4Hiss  and  Atkinson,  Jour.  Expcr.  Med.,  v,  1900. 


250  INFECTION   AND  IMMUNITY 

of  immunity  by  the  investigations  of  Nuttall,25  v.  Fodor,  Buchner, 
and  others,  who  not  only  demonstrated  the  power  of  normal  blood 
serum  to  destroy  bacteria,  but  also  showed  that  this  property  of  blood 
serum  became  diminished  with  age  and  was  destroyed  completely  by 
heating  to  56°  C.  The  thermolabile  substance  of  the  blood  serum 
possessing  this  power  was  called  by  Buchner,26  alexin. 

Soon  after  this  work,  Behring,  in  collaboration  with  Kitasato27  and 
Wernicke,28  in  1890  and  1892,  made  further  important  advances  in 
the  elucidation  of  the  immunizing  processes  by  showing  that  the  blood 
sera  of  animals  actively  immunized  against  the  toxins  of  diphtheria 
and  tetanus  would  protect  normal  animals  against  the  poisons  of 
these  diseases.  He  believed,  at  the  time  of  discovery,  that  such  sera 
contained  substances  which  had  the  power  of  destroying  the  specific 
toxins  which  had  been  used  in  the  immunization.  He  called  these 
bodies  antitoxins.  While  Behring 's  first  conception  of  actual  toxin 
destruction  soon  proved  to  be  erroneous,  his  discovery  of  the  presence 
in  immune  sera  of  bodies  specifically  antagonistic  to  toxins  was  soon 
confirmed  and  extended,  and  stands  to-day  as  an  established  fact. 

Ehrlich,29  soon  after  Behring 's  announcement,  showed  that  specific 
antitoxins  could  also  be  produced  against  the  poisons  of  some  of  the 
higher  plants  antiricin,  antikrotm,  antirobin),  and  Calmette30  pro- 
duced similar 'antitoxins  against  snake  poison  (antivenin).  Stimulated 
by  these  researches,  other  observers  have,  since  then,  added  exten- 
sively to  the  list  of  poisons  against  which  antitoxins  can  be  produced. 
Kempner31  has  produced  antitoxin  against  the  poison  of  Bacillus 
botulinus,  and  Wassermann,32  against  that  of  Bacillus  pyocyaneus. 
Antitoxin  has  been  produced  by  Calmette33  against  the  poison  of 
the  scorpion,  and  by  Sachs34  against  that  of  the  spider.  Thus  a  large 
number  of  poisons  of  animal,  plant,  or  bacterial  origin  have  been 
found  capable  of  causing  the  production  of  specific  antibodies  in  the 
sera  of  animals  into  which  they  are  injected. 


25  Nuttall,  Zeit.  f.  Hyg.,  1886. 

26  Buchner,  Cent.  f.  Bakt.,  i,  1889. 

27  Behring  und  Kitasato,  Deut.  med.  Woch.,  1890,  No.  49. 

28  Behring  und  Wernicke,  Zeit.  f.  Hyg.,  1892. 
^Ehrlich,  Dent.  med.  Woch.,  1891. 
^Calmette,  Compt.  rend,  de  la  soe.  de  biol.,  1894. 
31  Kempner,  Zeit.  f.  Hyg.,  1897. 

*•'  Wassermann,  Zeit.  f.  Hyg.,  xxii. 

83  Calmette,  Ann.  de  1  'inst.  Pasteur,  1898. 

34  Sachs,  Hofm.  Beit.,  1902. 


DEFENSIVE   FACTORS  OF   THE   ANIMAL  ORGANISM          251 

The  formation  of  antitoxins  directed  against  soluble  poisons,  how- 
ever, did  not  explain  the  immunity  acquired  by  animals  against 
bacteria  like  Bacillus  anthracis,  the  cholera  vibrio,  and  others  which, 
unlike  diphtheria  and  tetanus,  produced  little  or  no  soluble  toxin. 
It  was  evident  that  the  antitoxic  property  of  immune  blood  serum 
was  by  no  means  the  sole  expression  of  its  protective  powers.  Much 
light  was  shed  upon  this  phase  of  the  subject  by  the  discoveries  of 
Pfeiffer  in  1894,  who  worked  along  the  lines  suggested  by  the  inves- 
tigations of  Nuttall  and  Buchner.  Pfeiffer35  showed  that  when 
cholera  spirilla  were  injected  into  the  peritoneal  cavity  of  cholera- 
immune  guinea-pigs,  the  microorganisms  rapidly  swelled  up,  be- 
came granular,  and  often  underwent  complete  solution.  The  same 
phenomenon  could  be  observed  when  the  bacteria  were  injected  into 
a  normal  animal  together  with  a  sufficient  quantity  of  cholera-im- 
mune36 serum. 

This  process  he  observed  microscopically  by  abstracting,  from  time 
to  time,  a  small  quantity  of  the  peritoneal  exudate  and  studying  it 
in  hanging-drop  preparations.  The  reaction  was  specific  in  that  the 
destructive  process  took  place  to  any  marked  extent  only  in  the  case 
of  the  bacteria  employed  in  the  immunization. 

Metchnikoff,37  Bordet,  and  others  not  only  confirmed  Pfeiffer's 
observation,  but  were  able  to  show  that  the  lytic  process  would  take 
place  in  vitro,  as  well  as  in  the  animal  body.  The  existence  of  a 
specific  destructive  process  in  immune  serum  was  thus  established 
for  the  vibrio  of  cholera  and  soon  extended  to  other  microorganisms. 
The  constituents  of  the  blood  serum  which  gave  rise  to  this  destructive 
phenomenon  were  spoken  of  as  b  act  erioly  sins. 

Following  closely  upon  the  heels  of  Pfeiffer's  observation  came  the 
discovery  of  another  specific  property  of  immune  serum  by  Gruber  and 
Durham.38  These  workers  noticed  that  certain  bacteria,  when  brought 
into  contact  with  the  serum  of  an  animal  immunized  against  them, 
were  clumped  together,  deprived  of  motility,  and  firmly  agglutinated. 
They  spoke  of  the  phenomenon  as  agglutination  and  of  the  substances 
in  the  serum  giving  rise  to  it  as  agglutinins. 

The  list  of  antibodies  was  again  enlarged  by  Kraus,39  who  in  1897 


35  Pfeiffer,  Zeit.  f.  Hyg.,  xviii,   1894. 

36  Pfeiffer  und  Isaeff,  ibid. 

87  Metchnikoff,  Ann.  de  1  'inst.  Pasteur,  1895. 

38  Gruber  und  Durham,  Munch,  med.  Woch.,  1896. 

w  Kraus,  E.,  Wien.  klin.  Woch.,  32,  1897. 


252  INFECTION   AND   IMMUNITY 

showed  that  precipitates  were  formed  when  filtrates  of  cultures  of 
cholera,  typhoid,  and  plague  bacilli  were  mixed  with  their  specific 
immune  sera.  He  called  the  substances  which  bestowed  this  property 
upon  the  sera  precipitins. 

The  treatment  of  the  animal  body,  therefore,  with  bacteria  or  their 
products  gives  rise  to  a  variety  of  reactions  which  result  in  the 
presence  of  the  "antibodies"  described  above.  Extensive  investiga- 
tion has  shown,  however,  that  the  power  of  stimulating  antibody 
production  is  a  phenomenon  not  limited  to  bacteria  and  their  products 
alone.  Antitoxins,  we  have  already  seen,  may  be  produced  with  a 
variety  of  poisons  of  plant  and  animal  origin.  Sensitizing,  agglu- 
tinating and  precipitating  effects  may,  likewise,  be  produced  by  the 
use  of  a  large  number  of  different  substances.  Chief  among  these, 
because  of  the  great  aid  they  have  given  to  the  theoretical  investiga- 
tion of  the  phenomena  of  immunity,  are  the  red  blood  cells.  Bordet40 
and,  independently  of  him,  Belfanti  and  Carbone41  showed  in  1898 
that  the  serum  of  animals  repeatedly  injected  with  the  defibrinated 
blood  of  another  species  exhibited  the  specific  power  of  dissolving 
the  red  blood  corpuscles  of  this  species.  This  was  the  first  demonstra- 
tion of  "hemolysis" — a  phenomenon  which,  because  of  the  ease  with 
which  it  can  be  observed  in  vitro,  has  much  facilitated  investigation. 

The  knowledge  that  specific  "cytotoxins"  or  cell-destroying  anti-bodies 
could  be  produced  by  injection  of  red  blood  cells  naturally  suggested  the 
possibility  of  analogous  reactions  for  other  tissue  cells.  It  was  not  long, 
therefore,  before  Metchnikoff42  and,  independently  of  him,  Landsteiner43 
succeeded,  by  repeated  injections  of  spermatozoa,  in  producing  a  serum 
which  would  seriously  injure  these  specialized  cells.  Von  Dungern44  ob- 
tained similar  results  with  the  ciliated  epithelium  of  the  trachea.  Since 
then  a  host  of  cytotoxins  have  been  produced  with  the  cells  of  various 
organs  and  tissues.  Thus,  Neisser  and  Wechsberg45  produced  leucotoxin 
(leucocytes);  Delezenne,4G  neurotoxin  and  hepatotoxin;  Surmont,47  pan- 
creas cytotoxin;  and  Bogart  and  Bernard,48  suprarenal  cytotoxin. 


40  Bordet,  Ann.   de  1'inst.  Pasteur,   1898. 

41  Belfanti  et  Carbone,  Giornale  della  E.  Acatl.  di  Torino,  July,  1898. 

42  Metchnikoff,  Ann.  de  I'inst.  Pasteur,  189S. 

43  Landsteiner,  Cent.  f.  Bakt.,  i,  25,  1899. 

44  v.  Dungern,  Munch,  med.  Woch.,  1899. 
4*Neisser  und  Wechsberg,  Zeit.  f.  Hyg.,  xxxvi,  1901. 

"Delezenne,  Ann.  de  I'mst.  Past.  1900;  Compt.  rend,  de  1'acad.  des  sci.,  1900. 
"  Surmont,  Compt.  rend,  de  la  soc.  de  biol.,  1901. 
48  Bogart  et  Bernard,  ibid.,  1891. 


DEFENSIVE   FACTORS  OF  THE  ANIMAL  ORGANISM          253 

One  of  the  most  interesting  of  the  cytotoxins,  moreover,  is  nephrotoxin 
— produced  by  the  treatment  of  animals  with  injections  of  emulsions  of 
kidney  tissue. 

In  all  cases  it  was  supposed  by  those  first  working  with  these  bodies, 
that  the  injection  of  the  sera  of  animals  previously  treated  with  any 
particular  tissue  substance  would  produce  specific  injury  upon  the  organs 
homologous  to  the  ones  used  in  immunization.  It  need  hardly  be  pointed 
out  how  very  important  such  phenomena  would  be  in  throwing  light  upon 
the  degenerative  pathological  lesions  occurring  in  disease.  As  a  matter 
of  fact,  however,  sera  so  produced  have  been  shown  to  be  specific  for 
certain  organs  in  a  limited  sense  only.  The  question  of  specific  cytotoxins 
has  been  of  especial  importance  in  the  case  of  nephritis,  where  Ascoli 
and  Figari49  and  others  have  suggested  an  autonephrotoxin  as  the  basis 
of  the  pathology  of  this  disease.  In  the  hands  of  Pearce  and  others,  how- 
ever, the  strict  specificity  of  nephrotoxin  could  not  be  upheld  and  the 
subject  is  still  in  the  experimental  stage. 

Recent  experiments  by  Pearce50  suggest  that  at  least  a  part  of  the 
local  injury  to  organs  exerted  by  such  "cytotoxic"  sera  may  not  be 
due  to  a  specific  action  upon  the  organ  cells  so  much  as  upon  the  hema- 
glutinating  action  of  the  sera  causing  embolism  and  necrosis. 

It  is  a  fact  also  that  most  cytotoxic  sera  are  usually  hemolytic  as  well. 
It  is  not  easy  to  decide,  therefore,  how  much  of  the  action  upon  the 
organs  is  due  to  their  true  cytotoxic  properties  and  how  much  is  attributable 
to  the  concomitant  action  upon  blood  cells.  The  extravagant  hopes  at 
first  based  upon  cytotoxin  investigation,  especially  in  regard  to  the  problem 
of  malignant  tumors,  have  been  disappointed,  and  much  is  still  obscure  in 
regard  to  the  cytotoxins  which  calls  for  further  research. 

The  many  points  of  similarity  existing  between  bacterial  toxins 
and  digestive  ferments,  by  animal  inoculation,  suggested  to  several 
observers,  the  possibility  of  producing  antibodies  against  the  latter. 
As  a  result,  a  number  of  antiferments  have  been  obtained,  chief  among 
which  are  antilab  (Morgenroth51),  antipepsin  (Sachs52),  antisteapsin 
(Schiitze53),  and  antilactase  (Schiitze). 

The  stimulation  of  antibody  formation  in  the  sera  of  animals  is 
a  consequence,  therefore,  of  the  injection  of  a  large  variety  of  sub- 
stances— some  of  them  poisonous,  some  of  them  entirely  innocuous. 


49  Ascoli  and  Figari,  Borl.  klin.  Woch.,  1902. 

60  Pearce,  Jour.  Exper.  Mod.,  viii,  190(5. 

61  Morgenroth,  Cent.  f.  Bakt.,  1899. 
^Saclis,  Fort.  d.  Med.,  1902. 

™Schutze,  Deut.  med.  Woch.,  1904;   Zeit.  f.  Hyg.,  1905. 


254  INFECTION   AND  IMMUNITY 

The  substances  possessing  this  power  have  been  conveniently  named 
antigen  or  antibody -producers  by  German  writers.  The  term  antigen 
—though  etymologically  wrong,  nevertheless  is  convenient  and  has 
crept  into  general  usage.  It  signifies  simply  a  substance  which  can 
stimulate  the  production  or  formation  of  an  antibody.  Such  sub- 
stances, so  far  as  is  known,  belong  to  the  group  of  proteins  and  are 
derivatives  of  animal  or  plant  tissues.  Being  proteins,  all  antigens 
are  colloids.  Recently,  however,  some  crystalloidal  substances  have 
been  described  as  possessing  antigenic  properties. 


CHAPTER   XIII 

TOXINS    AND    ANTITOXINS 

The  Toxin-Antitoxin  Reaction. — Apart  from  the  therapeutic  pos- 
sibilities disclosed  by  the  discovery  of  antitoxins,  new  light  of  in- 
estimable value  was  thrown  by  these  observations  upon  the  biological 
processes  involved  in  immunization.  The  most  vital  problem,  of 
course,  which  immediately  thrust  itself  upon  all  workers  in  this 
field,  was  the  question  as  to  the  nature  of  the  reaction  in  which  toxin 
was  rendered  innocuous  by  antitoxin. 

The  simplest  conception  of  this  process  would  be  an  actual 
destruction  of  the  toxin  by  its  specific  antitoxin,  and  it  is  not  un- 
natural, therefore,  that  this  was  the  view  which,  for  a  short  time, 
found  favor  with  some  observers.  Roux,  and  more  particularly 
Buchner,1  however,  under  the  sway  of  cellular  pathology,  advanced 
the  opinion  that  the  antitoxins  in  some  way  influenced  the  tissue 
cells,  rendering  them  more  resistant  against  the  toxins.  Antitoxin, 
according  to  this  theory,  did  not  act  directly  upon  toxin,  but  affected 
it  indirectly  through  the  mediation  of  tissue  cells.  Ehrlich,2  on  the 
other  hand,  conceived  that  the  reaction  of  toxin  and  antitoxin  was 
a  direct  union,  analogous  to  the  chemical  neutralization  of  an  acid 
by  a  base — an  opinion  in  which  Behring  soon  joined  him. 

The  conception  of  toxin  destruction  received  unanswerable 
refutation  by  the  experiments  of  Calmette.3  This  observer,  working 
with  snake  poison,  found  that  the  poison  itself  (unlike  most  other 
toxins)  possessed  the  property  of  resisting  heat  even  to  100°  C., 
while  its  specific  antitoxin,  like  other  antitoxins,  was  delicately 
thermolabile.  He  noted,  furthermore,  that  non-toxic  mixtures  of 
the  two  substances,  when  subjected  to  heat,  regained  their  toxic 
properties.  The  natural  inference  from  these  observations  could 

1  Buchner,   ' '  Schutzimpfung, "   etc.,    in   Penzoldt   u.    Stinzing,   "Handbuch   d. 
spez.  Therap.  d.  Inf  ektkrank., ' '  1894. 

2  Ehrlich,  Deut.  med.  Woch.,  1891. 

3  Calmette,  Ann.  de  Pinst.  Past.,  1895. 

255 


250  INFECTION  AND  IMMUNITY 

only  be  that  the  toxin  in  the  original  mixture  had  not  been  destroyed, 
but  had  been  merely  inactivated  by  the  presence  of  the  antitoxin, 
and  again  set  free  after  destruction  of  the  antitoxin  by  heat.  A 
similar  observation,  made  soon  after  by  Wassermann4  and  in  the 
case  of  pyocyaneus  toxin  and  antitoxin,  fully  supported  the  results 
of  Calmette. 

An  ingenious  proof  of  the  direct  action  of  antitoxin  upon  toxin 
was  obtained  by  Martin  and  Cherry.5  It  was  found  by  them  that 
very  dense  filters,  the  pores  of  which  had  been  filled  with  gelatin, 
permitted  toxin  to  pass  through  under  high  pressure,  while  the 
presumably  larger  antitoxin  molecule  was  held  back.  Through  such 
filters  they  forced  toxin-antitoxin  mixtures,  under  a  pressure  of 
fifty  atmospheres,  at  varying  intervals  after  mixing.  They  found 
that,  if  filtered  immediately,  all  the  toxin  in  the  mixtures  came 
through,  but  that,  as  the  interval  elapsing  between  mixing  and  filtra- 
tion was  prolonged,  less  and  less  toxin  appeared  in  the  filtrate, 
until,  finally,  two  hours  after  mixing,  no  toxin  whatever  passed 
through  the  filter.  Besides  demonstrating  the  direct  action  of  anti- 
toxin upon  toxin,  this  work  of  Martin  and  Cherry  showed  that  the 
element  of  time  entered  into  the  toxin-antitoxin  reaction,  just  as 
it  enters  into  reactions  of  known  chemical  nature.  The  absolute  non- 
participation  of  the  living  tissue  cells  in  these  reactions  was  demon- 
strated by  Ehrlich  himself.  Kobert  and  Stillmarck6  had  shown  that 
ricin  possessed  the  power  of  causing  the  red  blood  cells  of  de- 
fibrinated  blood  to  agglutinate  in  solid  clumps,  a  reaction  which 
could  easily  be  observed  in  vitro.  Ehrlich,7  who  had  obtained  anti- 
ricin  in  1891  by  injecting  rabbits  with  increasing  doses  of  ricin, 
found  that  this  antibody  possessed  the  power  of  preventing  the 
hemagglutinating  action  of  ricin  in  the  test  tube.  By  a  series  of 
quantitatively  graded  mixtures  of  ricin  and  antiricin,  with  red  blood 
cells  as  the  indicator  for  the  reaction,  he  succeeded  in  proving  not 
only  that  the  toxin-antitoxin  neutralization  was  in  no  way  dependent 
upon  the  living  animal  body,  but  that  definite  quantitative  relations 
existed  between  the  two  substances  entirely  analogous  to  those 
which,  according  to  the  law  of  multiple  proportions,  govern  reac- 
tions between  different  substances  of  known  chemical  nature. 

4  Wassermann,  Zeit.  f .  Hyg.,  xxii,  1896. 

5  Martin  and  Cherry,  Proc.  Eoyal  Soc.,  London,  Ixiii,  1898. 

6  Kobert  und  Stillmarck,  Arb.  d.  phar.  Inst.  Dor,pat,  1889. 

7  Ehrlich,  Fort.  d.  Med.,  1897. 


TOXINS  AND  ANTITOXINS  257 

Similar  quantitative  results  were  subsequently  obtained  by  Stephens 
and  Myers8  for  cobra  poison  and  its  antitoxin,  by  Kossel9  for  the 
toxic  eel  blood  serum,  and  by  Ehrlich10  for  the  hemolytic  tetanus 
poison  known  as  tetanolysin. 

The  introduction  of  the  test-tube  experiment  into  the  investiga- 
tion of  these  reactions  permitted  of  much  more  exact  observations, 
and  by  this  means,  as  well  as  by  careful,  quantitatively  graded, 
animal  experiments,  the  further  facts  were  ascertained  that  toxin  and 
antitoxin  combined  more  speedily  in  concentrated  than  in  dilute 
solutions,  and  that  warmth  hastened,  while  cold  retarded,  the  reac- 
tion— observations11  which  in  every  way  seem  to  bear  out  Ehrlich 's 
conception  of  the  chemical  nature  of  the  process. 

Ehrlich 's  Analysis  of  Diphtheria  Toxin. — Shortly  after  the  dis- 
covery and  therapeutic  application  of  diphtheria  antitoxin,  it  be- 
came apparent  that  no  two  sera,  though  similarly  produced,  could 
have  exactly  the  same  protective  value.  It  was  necessary,  therefore, 
to  establish  some  measure  or  standard  by  which  the  approximate 
strength  of  a  given  antitoxin  could  be  estimated.  Von  Behring12 
attempted  to  do  this  for  both  tetanus  and  diphtheria  antitoxins  by 
determining  the  quantity  of  immune  sera  which,  in  each  case,  was 
needed  to  protect  a  guinea-pig  of  known  weight  against  a  definite 
dose  of  a  standard  poison.  He  ascertained  the  quantity  of  standard 
toxin-bouillon  which  would  suffice  to  kill  a  guinea-pig  of  250  grams, 
and  called  this  quantity  the  " toxin  unit."  This  unit  was  later' 
more  exactly  limited  by  Ehrlich,  who,  considering  the  element  of 
time,  stated  it  as  the  quantity  sufficient  to  kill  a  guinea-pig  of  the 
given  weight  in  from  four  to  five  days. 

Appropriating  the  terminology  of  chemical  titration,  v.  Behring 
spoke  of  a  toxin-bouillon  which  contained  one  hundred  such  toxin 
units  in  a  cubic  centimeter,  as  a  " normal  toxin  solution"  ("DTN1 
M250"),  and  designated  as  "normal  antitoxin"  a  serum  capable  of 
neutralizing,  cubic  centimeter  for  cubic  centimeter,  the  normal 
poison.13  A  cubic  centimeter  of  such  an  antitoxic  serum  was  suffi- 

8  Stephens  and  Myers,  Jour,  of  Path,  and  Bact.,  1898. 

9  Kossel,  Berl.  klin.  Woch.,  1898. 

10  Ehrlich,  Berl.  klin.  Woch.,  1898. 

11  Knorr,  Fort.  d.  Med.,  1897. 

12  v.  Behring,  Deut.  med.  Woch.,  1893. 

13  DTN1  M250  signifies :  D,  Diphtheria ;  TN1,  Normal  Toxin  solution ;  M2SO,  Meer- 
schweinchen  or  guinea-pig  weighing  250  grams. 


258  INFECTION  AND  IMMUNITY 

cient,  therefore,  to  neutralize  one  hundred  toxin  units,  and  was 
spoken  of  as  an  "antitoxin  unit.5"  In  the  experiments  of  v.  Behring, 
toxin  and  antitoxin  had  been  separately  injected.  Ehrlich14  im- 
proved upon  this  method  by  mixing  toxin  and  antitoxin  before  in- 
jection, thereby  obviating  errors  arising  from  differences  which  may 
have  existed  in  the  depth  of  injection  or  rapidity  of  absorption. 

In  order,  however,  that  any  such  method  of  standardization  of 
antitoxin  may  be  practically  applicable,  it  is  necessary  to  produce 
either  a  stable  toxin  or  an  unchangeable  antitoxin.  This  Ehrlich 
achieved  for  antitoxin  by  drying  antitoxic  serum  in  vacua  and  pre- 
serving it  in  the  dark,  at  a  low  temperature  and  in  the  presence 
of  anhydrous  phosphoric  acid.  By  the  use  of  such  a  stable  antitoxin, 
various  toxins  may  be  measured  and  other  antitoxic  sera  estimated 
against  these. 


Toxophorex 
/  I  <Jroup 


Body  Cell 

FIG.  30. — TOXIN  AND  BODY  CELLS. 

Given  thus  a  constant  antitoxin,  the  standardization  of  toxins 
would  be  a  comparatively  simple  matter  were  the  poison  obtainable 
in  a  perfectly  pure  state.  Unfortunately  for  the  ease  of  measure- 
ment, however,  this  is  not  the  case.  The  problem  is  rendered  difficult 
by  a  number  of  complicating  factors,  many  of  which  have  been 
brought  to  light  by  Ehrlich15  in  his  laborious  researches  into  the 
quantitative  relationship  between  the  two  reacting  bodies. 

As  previously  stated,  it  had  been  noted  by  Ehrlich  and  others 
that  toxin  solutions  would  deteriorate  with  time;  that  is,  a  toxin- 
bouillon  which  was  found  soon  after  production  to  contain,  say, 
eighty  toxin  units  in  each  cubic  centimeter,  would,  after  four  or 
five  months,  be  found  to  contain  but  forty  units  in  the  same  gross 
quantity.  It  had  lost,  therefore,  in  this  case,  just  one-half  of  its 
toxic  power.  In  spite  of  this  loss,  however,  Ehrlich  found  that  such 

14  Ehrlich,  Kossel  und  Wassermann,  Dent.  med.  Woch.,  1894. 
™  Ehrlich,  Klin.  Jahrbuch,  vi,  1897;  Deut.  med.  Woch.,  1898. 


TOXINS  AND  ANTITOXINS  259 

bouillon  had  retained  its  full  original  power  of  neutralizing  anti- 
toxin. If  the  reaction  was  purely  one  of  chemical  neutralization, 
there  seemed  to  be  but  one  explanation  of  this.  The  toxin  molecule 
must  contain  two  separate  atom  groups.  One  of  these  must  possess 
the  power  of  binding  antitoxin  and  be  stable;  this  he  designates 
as  the  "haptophore"  or  "anchoring"  group.  The  other,  the  one 
by  which  the  toxin  molecule  exerts  its  deleterious  action,  must  be 
more  easily  changed  or  destroyed;  this  he  calls  the  "toxophore" 
or  "poison"  group.  In  the  altered  toxin-bouillon  in  which  a  part 
of  the  poisonous  action  has  been  lost  while  the  antitoxin-neutralizing 
power  is  intact,  the  toxophore  group  of  some  of  the  toxin  must 
have  been  changed  or  destroyed.  Such  altered  toxin  he  speaks  of 
as  "toxoid." 

In  support  of  this  hypothesis  and  for  the  purpose  of  perfecting 
the  methods  of  standardization,  Ehrlich  was  led  to  determine,  for 
a  large  variety  of  specimens  of  diphtheria  toxin,  the  precise  quan- 
tity, in  cubic  centimeters,  which  was  necessary  to  neutralize  exactly 
one  unit  of  his  standard  antitoxin.  This  he  accomplished  by  making 
a  series  of  toxin-antitoxin  mixtures,  in  each  of  which  the  quantity 
ef  antitoxin  was  exactly  one  unit,  while  the  amount  of  toxin  was 
gradually  increased.  These  mixtures  were  injected  into  guinea-pigs 
of  250  grams  weight.  It  is  self-evident  that  in  such  an  experiment 
the  mixtures  containing  the  smaller  quantities  of  toxin  would  have 
no  effect  upon  the  guinea-pigs.  Soon,  however,  a  mixture  would  be 
reached  in  which  toxin  would  be  sufficiently  in  excess  of  antitoxin 
to  produce  the  symptoms  of  slight  poisoning,  as  evidenced  in  local 
edema,  rise  of  temperature,  etc.  The  largest  quantity  of  toxin  which 
could  be  added  without  producing  such  symptoms  was  then  regarded 
as  exactly  neutralizing  the  antitoxin  unit.  This  quantity  of  toxin 
Ehrlich  speaks  of  as  "Limes  zero"  (Limes  =  threshold)  or,  briefly, 

"L0." 

For  instance: 

One  antitoxin  unit  -f-  0.6  c.c.  toxin  ......  No  symptoms  of  poisoning. 

"          "         "         0.8  c.c  ...........  "         "          "         " 

"          "      .  "         0.9  c.c  ...........  "         "          "         " 

u          "         "          1.    c.c.   ,  "         "          ll         " 

1.1  c.c  ...........  Local  edema.    Paralysis  in  30  days. 

1.2  c.c  ...........  Death  in  10  days. 


In  this  example,  L0,  therefore,  equals  1  c.c. 


260  INFECTION  AND   IMMUNITY 

It  is  obvious,  therefore,  that  because  of  the  great  difficulty  in 
estimating  the  very  slightest  evidences  of  toxic  action  in  guinea-pigs, 
a  more  exact  method  of  standardizing  the  poisons  against  antitoxin 
would  be  to  determine  how  much  toxin  would  be  required  to  neu- 
tralize one  antitoxin  unit  and  still  be  sufficiently  in  excess  to  cause 
the  death  of  a  guinea-pig  of  250  grams  in  four  to  five  days.  This 
would  then  correspond  to  the  action  of  one  toxin  unit,  unmixed 
with  antitoxin.  A  priori  it  would  seem  that  this  value  (expressed  by 
Ehrlich  as  " Limes  death"  or  "L+")  must  simply  be  Lo  plus  one 
toxin  unit.  This,  however,  was  found  not  to  be  the  case.  Thus, 
in  the  example  given,  in  which  T  (the  toxin  unit— the  quantity  of 
the  bouillon  killing  a  guinea-pig  of  250  grams  in  four  to  five  days) 
was  equal  to  0.01  c.c.,  L0  (the  quantity  of  toxin  completely  neu- 
tralizing one  antitoxin  unit)  was  found  to  be  1  c.c.  or  100  T.  In 
this  same  poison,  however,  L+  (the  quantity  of  toxin  necessary  both 
to  neutralize  one  antitoxin  unit  and  yet  to  be  sufficiently  in  excess 
of  neutralization  to  kill  a  guinea-pig  of  250  grams  in  four  or  five 
days)  was  not  found  to  be  merely  L0  -[-IT;  but  on  actual  experi- 
ment proved  to  be  L0  -\-  101  T. 

Expressed  graphically,  the  conditions  may  be  stated  as  follows: 

T=  .01  c.c.  of  the  toxin  bouillon. 

L+    (neutral,  of  1  antitox,  unit  yet  killing  1  pig)   ==  2.01  c.c.  of  201  T. 

L0     (complete  neutral,  of  1  antitox.  unit)  =  1.       c.c.  or  100  T. 


Difference  =  1.01  c.c.  or  101  T. 

Ehrlich,  at  first,  endeavored  to  explain  this  surprising  phenomenon 
on  the  basis  of  toxoids.  He  argued  that  the  toxoids  formed  by 
deterioration  of  toxin  might  be  conceived  as  possessing  three  dif- 
ferent degrees  of  affinity  for  antitoxin.  If  their  affinity  for  anti- 
toxin were  equal  to,  or  more  marked  than,  that  of  the  toxin  itself, 
they  could  have  no  influence  upon  the  dose  L  .  If,  however,  their 
affinity  for  antitoxin  were  weaker  than  that  of  toxin,  each  fresh 
toxin  unit  added  to  the  dose  LQ  would,  first  uniting  with  antitoxin, 
replace  a  corresponding  quantity  of  these  nontoxic  substances  of 
weaker  affinity,  and  L+  would  be  reached  only  after  all  of  these 
"epitoxoids,"  as  Ehrlich  called  them,  had  been  replaced,  and  toxin 
became  free  in  the  mixture. 


TOXINS  AND   ANTITOXINS  261 

Thus,  in  analyzing  our  example,  we  have : 

100  tox.-antitox.  +  100  epitox.-antitox.  —  LQ; 

add  1  T,and  we  have  101  tox.-antitox.-f99  epitoxoid-antitoxin+1  epitoxoid  free ; 
add  101  T  and  we  have  200  toxin-antitoxin +100  epitoxoid  free  +  1  T  free=  I+- 

Two  facts,  however,  led  Ehrlich  to  abandon  the  opinion  that 
epitoxoid  was  merely  a  variety  of  toxoid.  He  found,  in  the  first 
place,  that  the  stated  relations  between  L0  and  L+  were  true  for 
perfectly,  fresh  toxin-bouillon  in  which  little  or  no  deterioration 
had  taken  place.  He  observed,  furthermore,  that  in  old,  altered 
toxin  bouillon,  while  T  was  very  much  affected,  the  quantity  needed 
to  kill  a  pig  constantly  increasing,  and  the  number  of  actual  fatal 
doses  in  L0  constantly  decreasing  (by  reason  of  toxoid  formation), 
L+  remained  practically  unchanged. 

Simply  stated,  this  means  that  the  epitoxoids  or  substances  which 
have  weaker  affinity  for  antitoxin  than  toxin  itself  are  already 
present  in  fresh  bouillon  and  are  not  increased  with  time.  For  this 
reason,  Ehrlich  has  separated  these  substances  from  toxoids.  He 
calls  them  "toxon"  and  believes  them  to  be,  like  toxin,  primary 
secretory  products  of  the  diphtheria  bacilli.  The  toxoids  themselves, 
Ehrlich  believes,  are  of  two  kinds,  those  with  a  stronger  affinity 
for  antitoxin  than  toxin  itself  (protoxoids),  and  those  whose  affinity 
for  antitoxin  is  equal  to  that  of  toxin.  These  latter  he  calls  "syn- 
toxoids. ' ' 

The  toxon  (epitoxoid  originally),  as  Ehrlich  believes,  has  a 
haptophore  or  " binding"  group  similar  to  that  of  toxin,  but  a  dif- 
ferent toxophore  or  "poisoning"  group.  Qualitatively  it  has  been 
shown  to  differ  from  toxin  in  that,  lacking  the  power  to  produce 
acute  symptoms,  it  causes  gradual  emaciation  and  paresis  in  animals. 

That  this  difference  in  the  poisonous  action  of  toxin  and  toxon 
is  not  merely  a  quantitative  difference,  referable  to  small  quantities 
of  toxin,  was  proved  by  Dreyer  and  Madsen,16  who  showed  that  if 
they  made  a  toxin-antitoxin  mixture  in  which  after  injection  the 
only  evidence  of  incomplete  neutralization  lay  in  the  emaciation  and 
final  paralysis  of  the  test  animals,  the  quantity  of  such  a  mixture 
could  be  increased  five-  and  tenfold,  without  producing  the  true 
toxin  symptoms  in  animals.  These  authors,  too,  claim  to  have  been 


18  Dreyer  mid  Madsen,  Zeit.  f .  Hyg.,  xxxvii,  1901. 


262  INFECTION   AND   IMMUNITY 

able  to  immunize  against  toxin  with  such  mixtures,  thereby  proving 
the  identity  of  the  haptophore  groups  of  the  two  substances.  The 
importance  of  this  observation  will  become  more  evident  in  con- 
nection with  the  section  on  the  "side-chain  theory." 

METHOD  OF  PARTIAL  ABSORPTION  OF  TOXIN. — Ehrlich17  has  gathered 
more  exact  data  in  support  of  his  views  from  what  he  terms  the 
"Method  of  Partial  Absorption"  of  toxin  by  antitoxin. 

In  order  to  understand  this  method  clearly,  it  is  necessary  to 
remember  that  Ehrlich18  believes  the  union  of  toxin  with  antitoxin 
to  take  place  according  to  the  chemical  laws  of  valency.  Just  as  in 
H20  oxygen  has  an  atomic  valency  of  2  for  hydrogen,  so,  in  the 
fully  neutralized  toxin-antitoxin  compound,  he  believes  antitoxin  to 
have  a  valency  of  200  for  toxin.  It  would  require,  according  to 
this,  200  T  or  toxin  molecules  to  satisfy  the  affinities  of  one  antitoxin 
molecule.19 

This  belief  is  based  upon  the  following  consideration:  In  de- 
termining the  LQ  dose,  or  fully  neutralized  toxin-antitoxin  union, 
Ehrlich,  as  well  as  Madsen,  found  that  the  number  of  T  units  con- 
tained in  such  a  dose  was  almost  regularly  a  factor  of  one  hundred, 
recurring  again  and  again  as  25,  33,  50,  75,  etc.  This  pointed  to 
more  or  less  regularity  in  the  deterioration  of  toxin  into  toxoid,  and 
to  a  more  or  less  regular  relation  of  toxin  to  toxon.  Now,  as  we 
have  seen  before,  if  we  could  procure  a  perfectly  pure  toxin,  the 
L0  dose  plus  one  toxin  unit  would  give  us  the  L+  dose;  that  is, 
one  toxin  unit  in  excess  of  full  neutralization  would  suffice  to  kill 
a  guinea-pig  of  250  grams  in  four  to  five  days.  Since  a  perfectly 
pure  toxin,  however,  has  not  been  obtainable  up  to  the  present 
time,  it  is  clear  that  the  number  of  pure  toxin  bonds  contained  in 
L  +  must  be  less  than  the  actual  number  of  neutralizing  units  in  the 
combination,  a  part  of  the  antitoxin  being  bound  by  toxon  and 
toxoid.  The  actual  values  obtained  for  the  number  of  T  units  in 
L+  has  never  exceeded  200,  and  has  usually  been  more  than  100, 
the  highest  value  ascertained  by  Madsen  being  160.  Given,  there- 
fore, a  combining  value  which,  being  a  multiple  of  one  hundred, 
is  often  more  than  one  hundred,  but  in  an  obviously  impure  state 
has  never  reached  200,  it  is  most  likely  that  200  represents  the 
actual  value  sought  for. 

-''Ehrlich,   ' '  Gesammelte   Arbeiten  zur   Immunitatsf orsch., "   Berlin,    1904. 

18  Ehrlich,  Deut.  med.  Woch.,  1898. 

19  Ehrlich,  ' '  Schlussbetrachtungen, ; '  Nothnagel's  System. 


TOXINS  AND  ANTITOXINS  263 

Assuming,  therefore,  upon  the  foregoing  considerations,  that  the 
valency  of  antitoxin  for  toxin  is  200,  Ehrlich  carries  out  his  experi- 
ments in  the  following  way : 

Given  a  toxin,  the  unit  (T)  of  which  is  0.024  c.c.,  he  first  deter- 
mines the  L .  dose  which,  tested  against  the  standard  antitoxin  unit, 
in  this  case  Is  2.05  c.c.  But  2.05  c.c.  =  85  T.  (or  2.05  -=-  .024)  units. 
By  mixing  the  L+  dose  of  toxin  and  antitoxin  in  such  a  way  that 
the  quantity  of  antitoxin  is  gradually  increased,  while  the  toxin 
remains  always  L+ ,  and  determining  upon  animals  the  amount  of 
free  toxin  contained  in  each  mixture,  the  following  table  may  be 
constructed  :20 

0  antitox.  unit  representing       0  valencies  +£+  =  85     free  T  units. 
.1       "          ll  "  20         "        -KL+  =  85       "    "     li 

.25     "          "  "  50         "        +£+  =  60       "    ll     " 

.8       "          "  "  160         "        +L+=10       "    tl     " 

.9       "          "  li  180         "        +L+  =     3.5    "    ll     " 

It  is  plain  that  the  substances  with  the  strongest  affinity  for  anti- 
toxin must  be  bound  first  by  the  antitoxin.  This  does  not  diminish 
the  toxic  value  of  the  mixture ;  and  these  are  the  protoxoids.  Next 
are  bound  syntoxoids  and  toxins,  and,  finally,  the  toxons.  It  is 
plain  that,  by  this  method,  the  constitution  of  any  given  toxin  may 
be  ascertained,  and  Ehrlich  has  constructed,  on  the  basis  of  these 
observations,  what  he  terms  his  toxin  spectrum.  Minor  differences 
of  toxicity  and  affinity  for  the  antibody  have  caused  him,  by  the 
partial  saturation  method  described,  still  further  to  divide  toxin 
into  proto-,  deutero-,  and  trito-toxin. 

His  spectra  graphically  describe  the  constitution  of  any  given 
toxic  bouillon  and  trace  its  deterioration  as  follows: 

Ehrlich 's  opinions  as  to  the  constitution  of  toxin  have  borne 
important  practical  fruit  in  allowing  him  to  develop  a  system  of 
standardization  of  anti-toxin  which  will  be  considered  in  the  next 
chapter,  but  his  theoretical  conceptions  as  described  above  are  not 
accepted  as  truly  representing  the  conditions  at  the  present  day. 
His  assumption  of  the  complexity  of  toxic  filtrates,  that  is,  toxin, 
the  various  toxoids,  and  toxons,  results  from  strict  adherence  to  the 
belief  that  the  reaction  is  analogous  to  that  taking  place  between 
strong  acids  and  strong  bases.  The  first  to  seriously  throw  doubt 
upon  his  theoretical  conceptions  011  the  basis  of  experiment,  were 
Arrhenius  and  Madsen. 

Example  taken  from  Ehrlich,  Deut.  med.  Woch.,  1898. 


264  INFECTION  AND  IMMUNITY 

Modern  theories  of  solution  maintain  that  substances  in  solution 
are  broken  up  into  their  atoms  or  atom-groups,  known  as  ions.  Thus, 
NaCl  in  solution  would  be  "dissociated"  into  its  Na  ion  and  its 
Cl  ion,  the  completeness  of  the  dissociation  depending  upon  the 
concentration  of  the  solution.  A  solution  of  NaCl,  therefore,  con- 
tains, according  to  this  view,  three  substances,  NaCl  undissociated 
and  free  ions  of  Na  and  Cl,  the  relative  quantities  of  the  three 
present  in  any  given  solution  being  calculable,  and  depend  upon 
a  law  known  as  the  law  of  mass-action  of  Guldberg  and  Waage. 
These  free  ions  are  the  elements,  "therefore,  which  are  active  in  the 
formation  of  further  chemical  combination.  When  a  strong  acid, 
in  solution,  acts  upon  a  base,  say  HC1  upon  ammonia  (NH3),  strong 
acid  having  the  property  of  quite  complete  dissociation  in  relatively 
concentrated  solutions,  little  or  no  ammonia  would  remain  unbound. 
A  weak  acid,  like  boric  acid,  however,  not  being  as  completely 
dissociated,  would  leave  some  ammonia  uncombined  even  after  more 
quantitatively  sufficient  bone  acid  had  been  added.  Arrhenius  and 
Madsen,  on  the  basis  of  careful  researches  into  the  reaction  between 
tetanolysin  and  its  antibody,  believe  that  toxin  and  antitoxin  possess 
weak  chemical  avidity  for  each  other,  their  interaction  being  com- 
parable to  that  taking  place  between  a  weak  acid  and  a  base.  Toxin- 
antitoxin  solutions,  therefore,  would  contain  the  neutral  compound, 
but  at  the  same  time  uncombined  toxin  and  antitoxin.  The 
qualities  which  Ehrlich  ascribes  to  toxon,  they  believe,  are 
due  to  the:  unbound  toxin  present  in  such  mixtures.  In  careful 
studies  in  which  they  inhibited  the  hemolytic  action  of  ammonia  by 
gradual  addition  of  boric  acid,  they  were  able  to  show  complete 
parallelism  between  the  conditions  governing  this  neutralization  and 
those  concerned  in  their  tetanus  experiments.  Their  explanation  has 
the  advantage  of  great  simplicity  over  that  of  Ehrlich 's  and  also 
the  fact  that  it  takes  into  account  the  laws  of  dissociation  which 
always  takes  place  in  solutions  in  which  the  union  01  two  substances 
occurs.  We  cannot  enter  into  the  matter  at  greater  length  in  this 
place,  and  must  refer  the  reader  to  more  extensive  works  on  im- 
munity.21 Objections  to  the  ideas  of  Arrhenius  and  Madsen  have 
been  brought  forward  by  Nernst,  Bordet  and  others,  largely  on  the 
basis  that  toxin-antitoxin  are  probably  colloidal  in  nature,  and  that 
the  laws  of  dissociation  in  colloidal  reactions  are  not,  as  yet,  clear. 

21  See  Zinsser,  Infection  and  Kesistanee,  MacMillan  &  Co.,  New  York,  1917; 
Bordet,  Immunite,  Masson,  Paris,  1920. 


TOXINS  AND  ANTITOXINS  205 

We  do  know,  however,  from  recent  work,  that  antigen-antibody 
complexes  can  dissociate,  and  the  work  of  Loeb  has  shown  that 
reactions  of  proteins  are,  after  all,  in  many  ways  strictly  analogous 
to  ordinary  chemical  reactions  between  less  complexly  constituted 
substances.  Moreover,  direct  experimentation,  such  as  that  of  Land- 
steiner  on  agglutinated  typhoid  bacilli,  Gay  and  Chickering's  work 
on  the  dissociation  of  antibody  from  precipitates,  Huntoon's  work  on 
the  dissociation  of  antibody  from  sensitized  pneumococcv  as  well 
as  observations  made  on  the  dissociation  of  diphtheria  toxin  from 
toxin-antitoxin  mixtures  in  connection  with  active  immunization  in 
diphtheria,  have  now  shown  pretty  definitely  that  dissociation  of 
these  substances  in  the  animal  body  and  in  the  test  tube  may  take 
place.  In  the  light  of  these  newer  researches  the  ideas  of  Arrhenius 
and  Madsen,  must  eventually  be  reexamined. 

Bordet,  Landsteiner  and  others  have  brought  out  another  point 
of  view  which  might  explain  the  quantitative  relations  which  exist 
in  toxin-antitoxin  mixtures  as  worked  out  by  Ehrlich.  They  dwell 
particularly  upon  the  analogy  of  these  reactions  with  colloidal  reac- 
tions, assuming  that  when  antitoxin  is  mixed  with  toxin  in  amounts 
insufficient  to  completely  neutralize,  the  units  of  antitoxin  are  not 
taken  up  by  a  corresponding  fraction  of  the  total  toxin  present, 
leaving  a  part  of  the  toxin  absolutely  free,  but  that,  on  the  contrary, 
the  antitoxin  is  equally  distributed  over  all  the  toxin  units  present, 
leaving  all  of  them  partially  saturated.  This  would  not  sharply 
neutralize  a  part  of  the  toxin,  leaving  another  part  entirely  free 
to  exert  its  activity,  but  would  partially  neutralize  all  the  toxin. 
It  is  more  or  less  analogous,  as  Bordet  brings  out,  to  different 
degrees  of  coloration  which  are  produced  when  starch  absorbs  vary- 
ing quantities  of  iodine.  They  compare  it  to  an  adsorption  phe- 
nomenon, rather  than  to  a  true  chemical  reaction. 

More  or  less  in  harmony  with  this  view  is  the  Dansyz  effect,22 
which  is  as  follows:  When  diphtheria  toxin  is  added  to  its  anti-toxin 
in  two  fractions,  a  definite  period  elapsing  between  the  addition  of 
the  first  and  the  second  fraction,  much  more  toxin  remains  free 
than  when  the  total  quantities  are  mixed  at  once.  This  view  has 
been  interpreted  in  Ehrlich 's  sense  by  Von  Dungern,  but  by  a  very 
forced  process  of  reasoning.  We  may  assume  with  considerable  con- 
fidence at  the  present  time,  that,  while  the  Ehrlich  method  of 

"Dansyz,  Ann.  <le  PInst.  Past.,  16,  1902. 


266  INFECTION   AND   IMMUNITY 

standardization  is  still  the  most  useful  basis  for  therapeutic  unit 
determination,  the  theoretical  considerations  upon  which  it  is 
founded  can  no  longer  be  accepted  as  conclusive. 

This,  however,  need  not  prevent  us  from  discussing  the  side- 
chain  theory  which  has  no  direct  relationship  to  his  views  on  toxins, 
but  offers  a  very  ingenious  explanation  for  the  mysterious  problem 
of  antibody  formation  and  specificity. 

The  Side-Chain  Theory. — We  have  seen  that  the  extensive  re- 
searches of  Ehrlich  into  the  nature  of  the  toxin-antitoxin  reaction 
led  him  to  believe  that  the  two  bodies  underwent  chemical  union, 
forming  a  neutral  compound.  The  strictly  specific  character  of  such 
reactions,  furthermore,  diphtheria  antitoxin  binding  only  diphtheria 
toxin,  tetanus  antitoxin  only  tetanus  toxin,  etc.,  led  him  to  assume 
that  the  chemical  affinity  between  each  antibody  and  its  respective 
antigen  depended  upon  definite  atom  groups  contained  in  each. 

Ehrlich23  had,  in  1885,  published  a  treatise  in  which  he  discussed 
the  manner  of  cell-nutrition  and  advanced  the  opinion  that  in  order 
to  nourish  a  cell,  the  nutritive  substance  must  enter  directly  into 
chemical  combination  with  some  elements  of  the  cell  protoplasm. 
The  great  number  and  variety  of  chemical  substances  which  act  as 
nutriment  led  him  to  believe  that  the  highly  complex  protoplasmic 
molecules  of  cellstwere  made  up  of  a  central  atom-group  (Leistungs- 
Kern)  upon  which  depended  the  specialized  activities  of  the  cell, 
and  a  multiplicity  of  side  chains  (a  term  borrowed  from  the  chem- 
istry of  the  benzol  group),  by  means  of  which  the  cell  entered  into 
chemical  relation  with  food  and  other  substances  brought  to  it  by 
the  circulation.  If  we  illustrate  graphically  by  the  chemical  concep- 
tion from  which  the  term  side  chain  was  borrowed,  in  salicylic  acid, 
the  formula  given,  the  benzol  ring  represents  the  "Leistungs-Kern," 

OH 


H— C      C— COOH 


H— C      C— H 


H 


23 Ehrlich,  "Das  Sauerstoffbediirfniss  des  Organismus, "  Berlin,  1885. 


TOXINS  AND  ANTITOXINS  2(57 

or  radicle,  while  COOH  and  OH  are  side  chains  by  means  of  which 
a  variety  of  other  substances  may  be  brought  into  relation  with  the 
"radicle,"  for  instance,  as  in  methyl  salicylate. 

OH 


C02CH3 


Just  as  nutritious  substances  are  thus  brought  into  workable 
relation  with  the  cell  by  means  of  the  atom-groups  corresponding 
to  side  chains,  so  Ehrlich  believes  toxins  exert  their  deleterious 
action  only  because  the  cells  possess  side  chains  by  means  of  which 
the  toxin  can  be  chemically  bound.  These  side  chains,  Ehrlich  in 
his  later  work  calls  "receptors."  The  receptors  or  side  chains 
present  in  the  cells  and  possessing  by  chance  specific  affinity  for  a 

Toxin. 
—Antitoxin 


fWWIff- 


FIG.  31. — TOXIN  AND  ANTITOXIN. 

given  toxin,  are,  by  their  union  with  toxin,  rendered  useless  for 
their  normal  physiological  function.  By  the  normal  reparative 
mechanism  of  the  body  these  receptors  are  probably  cast  off  and 
regenerated.  Regenerative  processes  of  the  body,  however,  do  not, 
as  a  rule,  stop  at  simple  replacement  of  lost  elements,  but,  according 
to  the  hypothesis  of  Weigert,24  usually  tend  to  overcompensation. 
The  receptors  eliminated  by  toxin  absorption  are  not,  therefore, 
simply  reproduced  in  the  same  quantity  in  which  they  are  lost,  but 
are  reproduced  in  excess  of  the  simple^  physiological  needs  of 
the  cell.  Continuous  and  increasing  dosage  with  the  poison,  conse- 
quently, soon  leads  to  such  excessive  production  of  the  par- 
ticular receptive  atom-groups  that  the  cells  involved  in  the 

24  Weigert,  Verhandl.  d.  Ges.  Deutsch.  Naturf .  u.  Aerzte,  Frankfurt,  189G. 


268  INFECTION  AND   IMMUNITY 

process  become  overstocked  and  cast  them  off  to  circulate  freely 
in  the  blood.  These  freely  circulating  receptors — atom-groups  with 
specific  affinity  for  the  toxins  used  in  their  production — represent 
the  antitoxins.  These,  by  uniting  with  the  poison  before  it  can  reach 
the  sensitive  cells,  prevent  its  deleterious  action. 

The  theory  of  Ehrlich,  in  brief,  then,  depends  upon  the  assump- 
tions that  toxin  and  antitoxin  enter  into  chemical  union,  that  each 
toxin  possesses  a  specific  atom-group  by  means  of  which  it  is  bound 
to  a  pre-existing  side  chain  of  the  affected  cell,  and  that  these  side 
chains,  in  accordance  with  Weigert's  law,  under  the  influence  of 
repeated  toxin  stimulation,  are  eventually  overproduced  and  cast  off 
by  the  cell  into  the  circulation. 

It  stands  to  reason  that  this  theoretical  conception  would  be 
vastly  strengthened  were  it  possible  to  show  that  such  receptors  or 
toxin-binding  atom-groups  actually  pre-existed  in  the  animal  body, 
and  such  support  was  indeed  given  by  the  experiments  of  Wasser- 
mann  and  Takaki.25  These  observers  succeeded  in  showing  that 
tetanus  toxin  could  be  rendered  innocuous  if,  before  injection  into 
animals,  it  was  thoroughly  mixed  with  a  sufficient  quantity  of  the 
fresh  brain  substance  of  guinea-pigs.  Similar  observations  were  in- 
dependently made  by  Asakawa,26  and  variously  confirmed.  Kempner 
and  Schepilewsky27  showed  a  similar  relation  to  exist  between  brain 
tissue  and  botulismus  toxin,  and  Myers28  brought  proof  of  analogous 
conditions  in  the  case  of  suprarenal  tissue  and  cobra  poison. 


25  Wassermann  und  Takaki,  Berl.  klin.  Woch.,  1898. 
MAsakawa,  Cent.  f.  Bakt,,  1898. 

27  Kempner  und  Schepileivsky,  Zeit.  f .  Hyg.,  1898. 

28  Myers,  Cent,  f .  Bakt.,  i,  1899. 


CHAPTER    XIV 

PRODUCTION  AND  TESTING  OF  ANTITOXINS 

DIPHTHERIA    ANTITOXIN 

IN  spite  of  the  great  advances  in  our  theoretical  knowledge  of 
antibodies,  gained  during  the  last  three  decades,  extensive  thera- 
peutic application  has  been  made  of  the  antitoxins  only.  Pre- 
eminent among  these  from  a  practical  point  of  view  are  the  anti- 
toxins against  diphtheria  and  tetanus  toxin.  For  diphtheria,  careful 
statistical  studies  have  demonstrated,  beyond  doubt,  the  therapeutic 
value  of  the  serum  treatment.  Biggs  and  Guerard,  in  a  general 
statistical  review,  arrived  at  the  conclusion  that  the  death  rate  of 
diphtheria  had  been  reduced  fifty  per  cent  by  the  use  of  antitoxin. 
Approximately  the  same  estimate  is  made  by  Dieudonne1  who  studied 
almost  10,000  treated  cases. 

Production  of  Diphtheria  Antitoxin. — The  methods  for  producing 
diphtheria  antitoxin  vary  only  in  minor  technical  details.  The  first 
requisite  for  successful  antitoxin  production  is  the  possession  of  a 
strong  toxin.  The  various  means  of  obtaining  this  are  outlined  in 
the  section  on  diphtheria  toxin.  The  toxin  used  should  be  of  such 
potency  that  less  than  0.1  c.c.  will  kill  a  guinea-pig  of  250  grams 
weight  in  four  to  five  days.2 

For  experimental  purposes,  goats  or  sheep  may  be  used  for 
immunization ;  for  antitoxin  production  on  a  large  scale,  horses  have 
been  found  to  be  the  most  useful  animals.  The  horses  should  be 
young,  four  to  six  years  old,  vigorous,  and  healthy.  It  is  advisable 
that  they  be  subjected  to  the  mallein  test  to  exclude  possible  infec- 
tion with  glanders. 

The  toxin  injections  are  made  subcutaneously.  Because  of  the 
differences  in  susceptibility  noted  in  various  horses,  it  is  advisable 
that  the  first  doses  of  toxin  should  be  either  very  small  or  weakened 


1  Dieudonne,  Arb.  a.  d.  kais.  Gesundheitsamt,  1895  and  1897. 
2 Park,  "Pathog.  Bacteria  and  Protozoa,"  N.  Y.,  1908. 

269 


270  INFECTION   AND   IMMUNITY 

by  chemicals  or  heat,  or  combined  with  antitoxin.  In  the  Pasteur 
Institute  in  Paris,  the  small  initial  dose  of  toxin  (0.5  c.c.)  is  mixed 
before  injection  with  an  equal  quantity  of  Lugol's  solution  (iodiii- 
potassium  iodid  solution). 

Park3  advises  an  initial  dose  of  5,000  toxin  units  (about  20  c.c. 
of  toxin)  combined  with  100  units  of  antitoxin.  The  same  amount 
is  given  with  the  second  and  third  doses  of  toxin.  The  intervals 
are  from  five  days  to  a  week,  determined  by  complete  subsidence  of 
the  reaction  (temperature).  The  doses  are  increased  until,  at  the 
end  of  two  or  three  months,  more  than  ten  times  the  original  dose 
is  given  (50,000  units). 

Horses  vary  greatly  in  the  strength  of  antitoxin  which  they  will 
produce.  At  the  end  of  three  or  four  months  in  favorable  animals 
one  cubic  centimeter  of  serum  may  contain  250  to  800  antitoxin 
units.  Further  immunization  will  often  increase  the  antitoxin  out- 
put to  1,000  and  more  units  to  the  cubic  centimeter  of  serum.  Park 
states  that  none  of  the  horses  used  by  him  has  ever  yielded  2,000 
units  to  the  cubic  centimeter.  The  same  writer  advises  a  three 
months'  period  of  rest  from  immunization  at  the  end  of  every  nine 
months.  Given  such  resting  periods,  some  horses  have  continued 
to  furnish  high-grade  antitoxin  for  from  two  to  four  years. 

In  order  to  obtain  serum  from  horses,  a  sharp  cannula  is  in- 
troduced into  the  jugular  vein.  After  leading  the  horse  into  a 
specially  constructed  stall,  its  head  is  slightly  deflected  and  pressure 
is  made  upon  the  jugular  vein  below  the  point  into  which  the  needle 
is  to  be  plunged.  Compression  can  also  be  made  by  surrounding  the 
neck  of  the  horse  close  to  the  shoulders  with  a  leather  strap  over 
a  pad  laid  directly  upon  the  vein.  The  vein  becomes  visible  along 
the  lower  margin  of  the  neck  in  a  line  running  from  the  angle  of 
the  jaw  to  the  edge  of  the  scapula.  The  skin,  of  course,  is  previously 
shaved  and  sterilized.  The  cannula  is  then  plunged  into  the  vein, 
either  with  or  without  previous  incision  through  the  skin,  and, 
through  a  sterile  rubber  tube,  the  blood  is  allowed  to  flow  into  high 
class  cylinders  or  slanted  Erlenmeyer  flasks.  In  this  way,  large 
quantities  of  blood  may  be  obtained  and,  according  to  Kretz,4  as 
much  as  six  liters  may  be  taken  at  a  time  at  intervals  of  a  month, 


'Park,  loc.  cit.,  p.  212. 

4  Kretz,  in  "Handb.   der   Techn.   u.   Meth.   d.  Immun., "   Kraus  and  Levaditi, 
vol.  ii,  1908. 


PRODUCTION   AND   TESTING   OF  ANTITOXINS  271 

without  injuring  the  animal.  Ligature  of  the  vein  after  bleeding 
is  unnecessary. 

The  cylinders  and  flasks  are  allowed  to  stand  for  two  or  three 
days  at  or  below  10°  C.  At  the  end  of  this  time,  the  serum  may 
be  pipetted  or  siphoned  away  from  the  clot  and  stored  in  the 
refrigerator.  In  order  to  diminish  the  chances  of  contamination, 
five-tenths  per  cent  of  carbolic  acid  or  four-tenths  per  cent  of  tri- 
eresol  may  be  added. 

Antitoxin  is  fairly  stable  and  if  kept  in  a  cool,  dark  place,  may 
remain  active,  with  but  slight  deterioration,  for  as  long  as  a  year. 
Kept  in  a  dry  state,  m  vacuo,  over  anhydrous  phosphoric  acid,  by 
the  method  of  Ehrlich,  it  retains  its  strength  indefinitely. 

Standardization. — Antitoxin  units  being  measured  in  terms  of 
toxin,  uniformly  of  measurement  necessitates  the  possession  by  the 
various  laboratories  of  a  uniform  toxin.  Antitoxin  being  more  stable 
than  toxin,  uniformity  of  toxin  is  obtained  by  means  of  a  standard 
antitoxin  distributed  from  a  central  laboratory.  This  was  first  done 
by  Ehrlich  in  Germany,  and  is  now  done  for  the  United  States  by 
the  Public  Health  and  Marine  Hospital  Service  laboratories.  Bottles 
of  the  distributed  antitoxin  are  marked  with  the  number  of  units 
contained  in  each  c.c.  Dilutions  of  this  are  mixed  with  varying 
quantities  of  the  toxin  to  be  tested,  the  mixtures  are  allowed  to 
stand  for  15  minutes  to  permit  union  of  the  two  elements,  and 
injections  into  guinea-pigs  of  250  grams  weight  are  made.  Thus  the 
L+  dose  of  the  toxin  is  determined.  (The  L+  dose  [p.  260]  is  the 
quantity  of  poison  not  only  sufficient  to  neutralize  one  antitoxin 
unit,5  but  to  contain  an  excess  beyond  this  sufficient  to  kill  a  guinea- 
pig  of  250  grams  in  4  to  5  days.  L+  is  chosen  rather  than  L0,  the 
simple  neutralizing  dose,  because  of  the  difference  between  toxins 
in  their  contents  of  toxoid  and  toxon.6) 

The  L+    dose  of  the  toxin  having  thus  been   determined,  this 

5  The  older  definition  of  a  unit  of  diphtheria  antitoxin  is  the  quantity  of  anti- 
toxin sufficient  to  protect  a  guinea-pig  of  250  grams  against  100  times  the  fatal 
dose  of  diphtheria  toxin.  This,  however,  holds  true  only  if  we  are  dealing  with 
normal  toxins  and  antitoxins  as  at  first  devised  by  Behring.  In  the  conditions 
under  which  the  measurements  are  made  at  present,  however,  this  definition  n.ust 
be  revised  as  follows:  A  unit  of  antitoxin  is  that  amount  of  antitoxin  which 
will  save  the  life  of  a  guinea-pig  if  injected  together  with  an  L  dose  of  the 
toxin. 

'  Madsen,  in  Kraus  u.  Levaditi,  "Handbuch/7  etc.,  1907. 


272  INFECTION  AND   IMMUNITY 

quantity  is  mixed  with  varying  dilutions  of  the  unknown  antitoxin.7 
Thus,  given  an  antitoxin  in  which  300  to  400  units  to  the  c.c.  are 
suspected,  dilutions  of  1:200,  1:250,  1:300,  etc.,  are  made.  One 
c.c.  of  each  of  these  is  mixed  with  the  L+  dose  of  the  toxin,  and 
the  mixtures  are  injected  into  guinea-pigs  of  about  250  grams.  If 
the  guinea-pig  receiving  L+  plus  the  1 :250  dilution  lives  and  the 
one  receiving  L+  plus  the  1 :300  dilution  dies  in  the  given  time,  we 
know  that  the  unit  sought  must  lie  between  these  two  values,  and 
further  similar  experiments  will  easily  limit  it  more  exactly.  The 
possibility  of  error  in  carrying  out  such  measurement  is  much 
diminished  by  the  use  of  larger  quantities  of  dilutions  higher  than 
those  given.  Four  c.c.  is  the  volume  usually  injected. 

Since  1902,  the  production  and  sale  of  diphtheria  antitoxin  has 
been  regulated  by  law  in  the  United  States.  From  time  to  time, 
antitoxin  is  bought  in  the  open  market  and  examined  at  the  hygienic 
laboratories  of  the  United  States  Public  Health  and  Marine  Hospital 
Service.  Antitoxic  serum  which  contains  less  than  two  hundred 
units  to  each  cubic  centimeter  is  not  permitted  upon  the  market. 

In  a  previous  section  we  have  seen  that  Hiss  and  Atkinson8  and 
others  have  shown  an  increase  in  the  globulin  contents  of  blood 
serum  of  immunized  animals.  It  has  been  shown,  furthermore, 
that  the  precipitation  of  such  serum  with  ammonium  sulphate  car- 
ried down  in  the  globulin  precipitate  all  the  antitoxic  substances 
contained  in  the  serum.  Upon  a  basis  of  globulin  precipitation, 
Gibson9  has  recently  perfected  a  method  of  concentrating  and  puri- 
fying diphtheria  antitoxin  for  therapeutic  use.  This  procedure,  as 
carried  out  at  the  New  York  Department  of  Health,  is,  in  principle, 
as  follows: 

The  serum,  as  taken  from  the  horse,  is  heated  to  56°  C.  for 
twelvr  hours.  This  converts  about  half  of  the  pseudoglobulin  into 
euglobulin,  the  antitoxin  remaining  in  the  pseudoglobulin  fraction.10 
It  is  then11  precipitated  with  an  equal  volume  of  a  saturated  am- 
monium sulphate  solution.  After  two  hours,  the  precipitate  is  caught 
in  a  filter  and  redissolved  in  a  quantity  of  water  corresponding  to 


T  Ddnits,  "Die  Werthbem.  der  Heilsera, "  in  Kolle  u.  Wassormann. 

87/is\s  and  Atkinson,  Jour.  Exper.  Med.,  v,  1900. 

8  Gibson,  Jour,  of  Biol.  Chem.,  i,  1906. 

111  Dr.  Banzltaf,  personal  communication. 

11  Gibson  and  Collins,  Jour,  of  Biol.  Chem.,  iii,  1907. 


PRODUCTION   AND  TESTING  OF  ANTITOXINS  273 

the  original  quantity  of  serum.  After  filtration,  this  solution  is 
again  precipitated  with  saturated  ammonium  sulphate  solution  and 
the  precipitate  again  filtered  off.  The  precipitate  is  then  treated 
with  a  saturated  solution  of  sodium  chloride  of  double  the  volume 
of  the  original  serum.  This  is  allowed  to  stand  for  about  twelve 
hours.  At  the  end  of  this  time  the  antitoxin-containing  globulin 
is  in  solution  and  is  pipetted  away  from  the  precipitate  and  filtered. 
This  salt-solution  extract  is  then  precipitated  with  twenty-five 
hundredths  per  cent  acetic  acid.  The  resulting  precipitate  of 
globulin  is  thoroughly  dried  by  pressure  between  filter  papers  and 
placed  in  a  parchment  dialyzer.  Dialysis  with  running  water  is 
continued  for  seven  to  eight  days,  after  neutralization  with  sodium 
carbonate,  in  order  to  remove  the  sodium  chloride.  At  the  end  of 
this  time,  the  globulin  solution  remaining  in  the  dialyzer  is  filtered 
through  a  Berkefeld  candle  for  the  purpose  of  sterilization,  after 
the  addition  of  0.8  per  cent  sodium  chlorid.  According  to  Gibson, 
this  method  produces  a  yield  of  antitoxin  which  equals  about  four- 
fifths  of  the  original  quantity  but  is  concentrated  five-  to  seven-fold. 
The  method  has  more  recently  been  modified  as  follows : 

After  heating  to  56°  C.,  as  above,  and  cooling,  ammonium  sul- 
phate is  added  to  the  serum  to  thirty  per  cent  saturation.  This 
brings  down  all  the  euglobulins.  This  is  then  filtered  and  the  filtrate, 
which  contains  the  pseudoglobulins  with  the  antitoxin,  is  again  pre- 
cipitated with  ammonium  sulphate  in  a  concentration  of  fifty-four 
per  cent  of  saturation.  The  precipitate  is  then  separated  on  a  paper, 
pressed  to  dryness,  and  directly  dialyzed.12 

Park  and  Thorne13  have  found  that  the  use  of  such  concentrated 
antitoxin  is,  therapeutically,  equally  efficient  as  the  unconcentrated, 
and  possesses  the  advantage  of  less  frequently  giving  rise  to  the 
secondary  reactions  in  skin  and  mucous  membranes  occasionally 
noticed  after  the  use  of  ordinary  antitoxin,  and  referable,  probably, 
to  some  other  constituent  of  the  horse  serum. 

Diphtheria  antitoxin  is  therapeutically  used  in  doses  ranging 
from  3,000  to  20,000  units.  For  prophylactic  immunization  of  healthy 
individuals,  about  500  units  should  be  used. 

See  also  the  chapter  on  diphtheria  for  Schick  reaction  and  active 
immunization  with  toxin-antitoxin  mixtures. 


12  Dr.  Banzhaf,  personal  communication. 

13Parfe  and  Thorne,  Amer.  Jour.  Med.  Sci.,  Nov.,  1906. 


274  INFECTION  AND  IMMUNITY 

TETANUS   ANTITOXIN 

Production  of  Tetanus  Antitoxin. — The  production  of  tetanus 
antitoxin  is,  in  every  way,  analogous  to  that  of  diphtheria  antitoxin. 
It  is  necessary  in  the  first  place  to  produce  a  powerful  tetanus  toxin. 
The  methods  of  procuring  this  will  be  discussed  in  the  section 
upon  tetanus  toxin.  Suffice  it  to  say  here  that  the  most  satis- 
factory method  of  obtaining  toxins  consists  in  cultivating  the  bacilli 
upon  veal  broth  containing  five-tenths  per  cent  to  two  per  cent 
sodium  chlorid  and  one  per  cent  pepton.  It  has  been  advised,  also, 
that  the  broth  should  be  neutralized  by  means  of  magnesium  car- 
bonate rather  than  with  sodium  hydrate.  The  bacilli  are  cultivated 
for  eight  to  ten  days  at  incubator  temperature  and  the  broth  filtered 
rapidly  through  Berkefeld  filters.  The  toxin  may  be  preserved  in 
the  liquid  form  with  the  addition  of  five-tenths  per  cent  carbolic 
acid,  or  may  be  preserved  in  the  dry  state  after  precipitation  with 
ammonium  sulphate. 

It  is  necessary  to  determine  the  strength  of  the  poison.  This  is 
done  according  to  v.  Behring14  by  determining  the  smallest  amount  of 
toxin  which  will  kill  a  white  mouse  of  twenty  grams  weight  within 
four  days.  This  is  most  easily  done  by  making  dilutions  of  the  toxin 
ranging  from  1 :100  to  1 :1,000,  and  then  injecting  quantities  of 
0.1  c.c.  of  each  of  these  dilutions  subcutaneously  into  white  mice. 
In  this  way,  the  minimal  lethal  dose  is  ascertained. 

For  the  actual  production  of  antitoxin,  horses  have  been  generally 
found  to  be  the  most  favorable  animals.  The  horses  should  be 
healthy  and  from  five  to  seven  years  old.  The  first  injection  of 
toxin  administered  to  these  animals  should  be  attenuated  in  some 
way.  Various  methods  for  accomplishing  this  have  been  in  use.  In 
America,  the  first  injection  of  about  ten  to  twenty  thousand  minimal 
lethal  doses15  (for  mice  of  twenty  grams  weight)  is  usually  made 
subcutaneously  together  with  sufficient  antitoxin  to  neutralize  this 
quantity.  In  Germany,  v.  Behring  uses,  for  his  first  injection,  a 
much  larger  dose  of  toxin  to  which  about  0.25  per  cent  of  terchlorid 
of  iodin  has  been  added.  Immediately  after  an  injection,  the  animals 
will  usually  show  a  reaction  expressed  by  a  rise  of  temperature, 

14  v.  Behring,  Zeit.  f .  Hyg.,  xii,  1892 ;  Deut.  med.  Woch.,  1900. 

15  According  to  Park  the  "horses  receive  5  c.c.  as  the  initial  dose  of  a  toxin 
of  which  1  c.c.  kills  250,000  grams  of  guinea-pig,  and  along  with  this  a  sufficient 
amount  of  antitoxin  to  neutralize  it. ' ' 


PRODUCTION   AND  TESTING  OF  ANTITOXINS  275 

refusal  of  food,  and  sometimes  muscular  twitching.  A  second  injec- 
tion should  never  be  given  until  all  such  symptoms  have  completely 
subsided.  This  being  the  case,  after  five  to  eight  days  double  the 
original  dose  is  given  together  with  a  neutralizing  amount  of  anti- 
toxin or  with  the  addition  of  terchlorid  of  iodin.  Again  after  five 
to  eight  days,  a  larger  dose  is  given  and  thereafter,  at  similar 
intervals,  the  quantity  of  toxin  is  rapidly  increased.  In  America 
the  neutralizing  antitoxin  is  omitted  after  the  third  or  fourth  injec- 
tion; in  v.  Behring's  laboratory  the  quantity  of  terchlorid  of  iodin 
is  gradually  diminished.  The  increase  of  dosage  is  often  controlled 
by  the  determination  of  the  antitoxin  contents  of  the  animal's  blood 
serum.  The  immunization  is  increased  until  enormous  doses  (500 
c.c.)  of  a  toxin  in  which  the  minimal  lethal  dose  for  mice  is  repre- 
sented by  0.0001  c.c.,  or  less,  is  borne  by  the  horse  without  apparent 
harm. 

The  antitoxic  serum  is  then  obtained  by  bleeding  from  the  jugular 
vein,  as  in  the  case  of  diphtheria  antitoxin.  It  may  be  preserved 
in  the  liquid  state  by  the  addition  of  five-tenths  per  cent  of  carbolic 
acid  or  four-tenths  per  cent  of  tricresol. 

Standardization. — The  universal  prophylactic  use  of  tetanus  anti- 
toxin has,  as  in  the  case  of  diphtheria  antitoxin,  necessitated  its 
standardization.  A  variety  of  methods  are  in  use  in  different  parts 
of  the  world.  In  the  following  description  the  American  method 
only  will  be  considered  as  laid  down  under  the  law  of  July,  1908, 
and  based  upon  the  work  of  Rosenau  and  Anderson16  at  the"  United 
States  Hygienic  Laboratories  at  Washington. 

In  conjunction  with  a  committee  of  the  Society  of  American 
Bacteriologists,  these  authors  have  defined  the  unit  of  tetanus  anti- 
toxin as  follows: 

The  unit  shall  be  ten  times  the  least  amount  of  serum  necessary 
to  save  the  life  of  a  350  gram  guinea-pig  for  ninety-six  hours  against 
the  official  test  dose  of  standard  toxin.  The  test  dose  consists  of 
100  minimal  lethal  doses  of  a  precipitated  toxin  preserved  under 
special  conditions  at  the  hygienic  laboratory  of  the  Public  Health 
and  Marine  Hospital  Service.  (The  minimal  lethal  dose  is  in  this 
case,  unlike  v.  Behring's  minimal  lethal  dose,  measured  not  against 
20  gram  mice,  but  against  350  gram  guinea-pigs.) 

In  the  actual  standardization  of  tetanus  antitoxin,  as  in  that  of 

18  Rosenau  and  Anderson,  Pub.  Health  and  Mar.  Hosp.  Serv.  TJ.  S.,  Hyg.  Lab. 
Bull.  43,  1908. 


276 


INFECTION   AND   IMMUNITY 


diphtheria  antitoxin,  the  L+  dose  of  toxin  is  employed.  The  L+ 
dose  is,  however,  in  this  case,  denned  as  the  smallest  quantity  of 
tetanus  toxin  that  will  neutralize  one-tenth  of  an  immunity  unit, 
plus  a  quantity  of  toxin  sufficient  to  kill  a  350  gram  guinea-pig 
in  just  four  days.  At  the  Hygienic  Laboratory  at  Washington,  a 
standard  toxin  and  antitoxin  are  preserved  under  special  conditions, 
and  standard  toxin  and  antitoxin,  arbitrary  in  their  first  establish- 
ment, are  kept  constant  by  being  measured  against  each  other  from 
time  "to  time.  In  measuring  the  antitoxic  serum  thus  preserved,  at 
the  Hygienic  Laboratory,  a  mixture  of  one-tenth  of  a  unit  of  anti- 
toxin and  100  minimal  lethal  doses  of  the  standard  toxin  must 
contain  just  enough  free  poison  to  kill  the  guinea-pig  in  four  days. 
This  L+  dose  of  the  standard  toxin  is  given  out  to  those  interested 
commercially  or  otherwise  in  the  production  of  antitoxin. 

In  measuring  an  unknown  antitoxic  serum  against  this  L+  dose 
of  toxin,  a  large  number  of  mixtures  are  made,  each  containing  the 
L+  dose  of  the  toxin  and  varying  quantities  of  the  antitoxin.  Dilu- 
tions must  always  be  made  with  0.85  per  cent  salt  solution  and  the 
total  quantity  injected  into  the  animals  should  always  be  brought  up 
to  4  c.c.  with  salt  solution  in  order  to  equalize  the  conditions  of 
concentration  and  pressure.  The  mixtures  are  then  kept  for  one 
hour  at  room  temperature  in  diffused  light.  After  this  they  are 
subcutaneously  injected  into  a  series  of  guinea-pigs  weighing  from 
300  to  400  grams.  The  following  example  of  a  test  is  taken  from 
the  article  by  Rosenau  and  Anderson  quoted  above. 


No.  of 
Guinea- 
pig. 

Weight  of 
Guinea-pig 

(Grams). 

SUBCUTANEOUS  INJECTION  OF  A 
MIXTURE  OF 

Time  of  Death. 

Toxin 
(Test  Dose). 

Antitoxin. 

1  

360 
350 
350 
360 
350 

Gram. 

0.0006 
.0006 
.0006 
.0006 
.0006 

c.c. 
0.001 

.0015 
.002 
.0025 
.003 

2  days  4  hours 
4  days  1  hour 
Symptoms 
Slight  symptoms 
No  symptoms 

2  
3  

4   . 

5 

In  this  series  the  guinea-pig,  receiving  0.0015  c.c.  of  the  antitoxin, 
died  in  approximately  four  days;  0.0015  c.c.  therefore  represents 
one-tenth  of  an  immunity  unit. 

In  therapeutically  employing  antitoxin  for  prophylactic  purposes, 
above  1,500  units  should  be  employed. 


CHAPTER   XV 

SENSITIZING    ANTIBODIES.   '  (PHENOMENA    OF    LYSIS,    AGGLUTINA- 
TION, PRECIPITATION,  E^C.) 

Alexin  and  Sensitizing  Antibodies. — In  the  immediately  preced- 
ing sections,  we  have  dealt  solely  with  immunity  as  it  occurs  where 
soluble  toxins  play  an  important  part  and  in  which  antitoxins  are 
developed  in  the  immunized  subject.  There  are  many  species  of 
pathogenic  bacteria,  however,  which  stimulate  the  production  of 
little  or  no  antitoxic  substance  when  introduced  into  animals,  and 
the  resistance  of  the  immunized  animal  can  not,  therefore,  be  ex- 
plained by  the  presence  of  antitoxin  in  the  blood. 

v.  Fodor,1  Nuttall,2  Buchner,3  and  others  had  in  1886  and  the 
years  following,  carried  on  investigations  which  showed  that  normal 
blood  serum  possessed  the  power  of  killing  certain  of  the  pathogenic 
bacteria.  Nuttall,  working  under  the  direction  of  Fliigge,  made 
the  important  discovery  that  this  bactericidal  power  became  grad- 
ually diminished  with  time,  and  could  be  experimentally  destroyed 
by  exposure  of  the  serum  to  a  temperature  of  56°  C.  for  one-half 
hour.  Buchner,  who  confirmed  and  extended  the  observations  of 
Nuttall,  called  this  thermolabile  substance  upon  which  the  bacteri- 
cidal character  of  the  serum  seemed  to  depend  "alexin. " 

Our  knowledge  of  the  bactericidal  action  of  serum  was,  soon 
thereafter,  extensively  increased  by  the  discovery,  by  Pfeiffer  and 
Isaeff,4  that  cholera  spirilla  injected  into  the  peritoneal  cavity  of 
a  cholera-immune  guinea-pig  were  promptly  killed  and  almost  com- 
pletely dissolved.  The  same  phenomenon  could  be  observed  when 
the  spirilla,  mixed  with  fresh  immune  serum,  were  injected  into  the 
peritoneum  of  a  normal  guinea-pig. 

The  processes  observed  by  Pfeiffer  as  taking  place  intraperi- 


*«.  Fodor,  Dent.  mod.  Woch.,  1886. 

2  Nuttall,  Zeit.  f.  Hyg.,  1886. 

1  Buchner,  Cent.  f.   Kakt.,    1SS9. 

4  Pfeiffer  und  Isaeff,  Zeit,  f.  Hyg.,  1894. 

277 


278  INFECTION   AND   IMMUNITY 

toneally  were  soon  shown  by  Metchnikoff,5  Bordet,6  and  others  to 
take  place,  though  to  a  lesser  extent,  in  vitro.  Bordet,  furthermore, 
observed  that  the  bacteriolytic  digestive  power  of  such  immune  serum, 
when  destroyed  by  heating,  or  after  being  attenuated  by  time,  could 
be  restored  by  the  addition  to  it  of  small  quantities  of  normal  blood 
serum.  It  could,  in  other  words,  be  "reactivated"  by  normal  serum. 
Prom  this  observation  Bordet  drew  the  conclusion  that  the  bacteri- 
cidal or  bacteriolytic  action  of  the  serum' depended  upon  two  distinct 
substances.  The  one  present  in  normal  serum  and  thermolabile,  he 
conceived  to  be  identical  with  Buchner's  alexin.  The  other,  more 
stable,  produced  or  at  least  increased  in  the  serum  by  the  process 
of  immunization,  he  called  the  "sensitizing  substance."  This  sub- 
stance, he  believed,  acting  upon  the  bacterial  cells,  rendered  them 
vulnerable  to  the  action  of  the  alexin.  Without  the  previous  prepara- 
tory action  of  the  "sensitizing  substance"  the  alexin  was  unable 
to  act.  Without  the  cooperation  of  alexin,  the  ' i  sensitizing  sub- 
stance" produced  no  visible  effects. 

Bordet 's  interpretation  of  the  phenomenon  of  lysis  differs  essen- 
tially from  that  of  Ehrlich,  in  that  both  active  serum  components  are 
conceived  by  him,  though  independent,  to  act  directly  upon  the 
bacterial  cell.  A  few  years  later,  Bordet  was  able  to  show  that 
exactly  analogous  conditions  governed  the  phenomenon  known  as 
"hemolysis"  or  disintegration  of  red  blood  cells. 

It  had  been  known  for  many  years  that  in  the  transfusion  of 
blood  from  an  animal  of  one  species  into  an  animal  of  another 
species,  injury  was  done  to  the  red  corpuscles  which  were  intro- 
duced. Observed  in  the  test  tube,  the  red  cells  in  the  heterologous 
serum  were  seen  to  give  up  their  hemoglobin  in  the  fluid,  the  mix- 
ture taking  on  the  red  transparency  characteristic  of  what  is  known 
as  "laked"  blood.  Buchner,7  in  his  alexin  studies,  had  shown  that 
the  blood-cell  destroying  action  of  the  normal  serum  was  subject 
to  the  same  laws  as  the  bactericidal  power  of  similar  serum,  in 
that  it  was  destroyed  by  heating,  and  he  assumed  that  both  the 
bacteriolytic  and  the  hemolytic  action  of  normal  serum  were  due  to 
the  same  "alexin."  Metchnikoff,8  moreover,  had  pointed  out  the 
striking  analogy  between  the  two  phenomena  as  early  as  1889. 


5  Metchnikoff.  Ann.  de  1 'inst.  Pasteur,  1895. 

9  Bordet,  ibid.,  1895. 

''Buchner,  Arch.  f.  Hyg.,  xvii,  1893;   Waremberg,  Arch.  d.  med.  exper.,  1891. 

8  Metchnikoff,  Ann.  de  1  'inst.  Pasteur,  1889. 


SENSITIZING  ANTIBODIES  279 

Bordet9  now  observed  that  the  blood  serum  of  guinea-pigs 
previously  treated  with  the  defibrinated  blood  of  rabbits  developed 
marked  powers  of  dissolving  rabbits'  corpuscles,  and  that  this 
hemolytic  action  could  be  destroyed  by  heating  to  56°  C.,  but 
" reactivated"  by  the  addition  of  fresh  normal  serum.  He  had  thus 
produced  an  immune  hemolysin,  just  as  Pfeiffer  had  produced  im- 
mune bacteriolysin,  and  had  demonstrated  the  complete  parallelism 
which  existed  between  the  two  phenomena. 

A  practical  test-tube  method  was  thus  given  for  the  investigation 
of  the  lysins,  just  as  a  practical  test-tube  method  for  antitoxin 
researches  had  been  developed  by  Ehrlich  in  his  ricin-antiricin  ex- 
periments. 

The  path  of  investigation  thus  pointed  out  by  Bordet  was  soon 
explored  in  greater  detail  by  Ehrlich  and  Morgenroth.10  The  rea- 
soning which  Ehrlich  had  applied  in  explaining  the  production  of 
antitoxins  was  thought,  by  these  observers,  to  be  equally  applicable 
to  the  phenomena  of  bacteriolysis  and  hemolysis. 

Since  the  thermolabile  substance  or  alexin,  renamed  by  Ehrlich 
"complement,"  was  already  present  in  normal  serum  and  had  been 
shown  to  be  little,  if  at  all,  increased  during  the  process  of  im- 
munization, this  substance  could  have  but  little  relation  to  the 
changes  taking  place  in  the  animal  body  as  immunity  was  acquired. 
The  more  stable  serum-component,  however,  the  "substance  sen- 
sibilisatrice "  of  Bordet,  or,  as  Ehrlich  now  called  it,  the  "immune 
body,"  was  the  one  which  seemed  specifically  called  forth  by  the 
process  of  active  immunization.  Ehrlich  argued,  therefore,  that 
when  bacteria  or  blood  cells  were  injected  into  the  animal,  certain 
atom-groups  or  chemical  components  of  the  injected  substances  were 
united  to  other  atom-groups  or  "side  chains"  of  the  protoplasm 
of  the  tissue  cells.  These  "side  chains"  or  receptors,  then  repro- 
duced in  excess  and  finally  thrown  free  into  the  circulation,  con- 
stituted the  "immune  body."  The  immune  body,  therefore,  he  con- 
cluded, must  possess  atom  complexes  which  endow  it  with  specific 
chemical  affinity  for  the  bacteria  or  red  blood  cells  used  in  its 
production.  This  contention  was  supported  by  Ehrlich  and  Mor- 
genroth by  an  ingenious  series  of  experiments. 

Having  in  their  possession,  at  that  time,  the  blood  serum  of  a 

9  Bordet,  Ann.  de  1'inst.  Pasteur,  t.  xii,  1898. 

10  Ehrlich  und  Morgenroth,  Berl.  klin.  Woch.,  1,  1899. 


280  INFECTION    AND    IMMUNITY 

goat  immunized  against  the  red  blood  cells  of  a  sheep,  they  inac- 
tivated it  (destroyed  the  complement  or  alexin)  by  heating  to  56° 
C.  The  serum  then  contained  only  the  ''substance  sensibilisatrice  " 
or  immune  body.  To  this  inactivated  serum  they  added  sheep's 
red  corpuscles,  without  obtaining  hemolysis.  Having  left  the  in- 
active serum  and  the  sheep's  corpuscles  in  contact  with  each  other 
for  some  time,  they  separated  them  by  centrifugalization.  To  the 
supernatant  fluid,  they  now  added  sheep-blood  corpuscles  and 
normal  goat  serum  (complement)  and  found  that  no  hemolysis  took 
place.  The  immune  body  had  apparently  gone  out  of  the  serum. 
The  red  cells  which  had  been  in  contact  with  the  serum  and  separated 
by  the  centrifuge  were  then  washed  in  salt  solution  and  to  them 
complement  was  added  in  the  form  of  fresh  normal  serum.  Hemolysis 


Substance    Complement 


Body  Cell 

FIG.  32. — EHRLICH'S  CONCEPTION  OF  CELL-RECEPTORS,  GIVING  RISE  TO  IMMUNE 

BODIES. 

occurred.  It  was  plain,  therefore,  that  the  immune  body  of  the 
inactivated  serum  had  gone  out  of  solution  and  had  become  attached 
to  the  red  blood  cells,  or,  as  Ehrlich  expressed  it,  the  immune  body 
by  means  of  its  '  *  haptophore "  atom-group  had  become  united  to  the 
corpuscles.  In  contrast  to  this,  if  normal  goat  serum  (containing 
complement  only)  was  added  to  sheep  corpuscles  and  separated  again 
by  centrifugalization,  the  supernatant  fluid  was  found  to  be  still 
capable  of  reactivating  inactivated  serum  (immune  body).  This  he 
interpreted  as  proving  that  the  complement  was  not  bound  to  the 
corpuscles  directly. 

If  the  three  factors  concerned — corpuscles,  immune  body,  and 
complement — were  mixed  and  the  mixture  kept  at  0°  C.,  no  hemolysis 
occurred;  yet,  centrifugalized  at  this  temperature,  immune  body 
was  found  to  have  become  bound  to  the  corpuscles,  the  complement 
remaining  free  in  the  supernatant  fluid.  If  the  same  mixture,  how- 
ever, was  exposed  to  37°  C.,  hemolysis  promptly  occurred. 


SENSITIZING  ANTIBODIES  281 

From  this,  Ehrlich  concluded  that  complement  did  not  directly 
combine  with  the  corpuscles,  but  did  so  through  the  intervention  of 
the  immune  body.  This  immune  body,  he  reasoned,  possessed  two 
distinct  atom-groups  or  haptophores;  one,  the  cytophile  haptophore 
group,  with  strong  affinity  for  the  red  blood  cell;  the  other,  or 
complementophile  haptophore  group,  with  weaker  avidity  for  the 
complement.  Because  of  this  double  combining  power,  Ehrlich 
speaks  of  the  immune  body  as  "amboceptor."  These  views  are 
graphically  represented  in  Figs.  132  and  133. 

Thus,  according  to  Ehrlich  and  his  pupils  the  alexin,  or  comple- 
ments, acts  upon  the  antigen  indirectly  only  through  the  "zwischen- 
koerper"  or  "amboceptor. "  These  views  of  Ehrlich  have  been 
described  in  more  or  less  detail  because  they  form  an  excellent 

Complement 

body 


Cell  used 


FIG.  33.  —  COMPLEMENT,  AMBOCEPTOR  OR  IMMUNE  BODY.  AND  ANTIGEN  OR 
IMMUNIZING  SUBSTANCE. 

introduction  to  a  study  of  these  phenomena.  We.  do  not  believe, 
however,  that  they  are  tenable  at  the  present  time.  It  was  Bordet 
particularly  who  has  pointed  out  many  of  the  uncertainties  of  the 
amboceptor  point  of  view.  The  only  thing  that  we  can  say  with 
certainty  is  that  alexin  or  complement  do  not  go  into  union  with 
the  unsensitized  antigen,  but  that  the  action  of  the  alexin  upon 
the  antigen  is  made  possible  only  by  preliminary  sensitization,  or, 
in  other  words,  by  union  with  the  specific  antibody.  This  complex 
then  is  amenable  to  alexin  action.  It  is  neither  necessary,  nor  is 
it  justifiable  on  the  basis  of  experimental  fact,  to  assume  that  the 
sensitizing  antibody  is  an  amboceptor  or  a  sort  of  bridge  between 
the  antigen  and  the  alexin.  The  difference  is  a  fundamental  one, 
and  all  the  facts  are  on  the  side  of  the  Bordet  view,  as  far  as  we 
can  see.  It  is  better  to  refer  to  the  substances  involved  as  sensitizer 
and  alexin,  instead  of  as  amboceptor  and  complement.  As  we  shall 


282  INFECTION   AND   IMMUNITY 

see,  we  believe  that  the  sensitizing  antibody,  whether  in  the  process 
of  lysis,  agglutination,  precipitation,  etc.,  is  in  all  cases  the  same 
substance. 

Agglutination. — Although  Metchnikoff11  and  Charrin  arid  Roger12 
had  noticed  peculiarities  in  the  growth  of  bacteria  when  cultivated 
in  immune  sera,  which  were  unquestionably  due  to  agglutination, 
the  first  recognition  of  the  agglutination  reaction  as  a  separate 
function  of  immune  sera  was  the  achievement  of  Gruber  and  Dur- 
ham. While  investigating  the  Pfeiffer  reaction  with  B.  coli  and 
the  cholera  vibrio,  Gruber  and  Durham13  noticed  that  if  the  respec- 
tive immune  sera  were  added  to  bouillon  cultures  of  these  two 
species,  the  cultures  would  lose  their  turbidity  and  flake-like  clumps 
would  sink  to  the  bottom  of  the  tube,  the  supernatant  fluid  becom- 
ing clear.  Gruber,  at  the  same  time,  called  attention  to  the  fact 
that  immune  sera  would  affect  in  this  way  not  only  the  microor- 
ganism used  in  their  production,  but,  to  a  less  energetic  extent, 
other  closely  related  bacteria  as  well. 

Widal,  very  soon  after  Gruber  and  Durham's  announcement, 
applied  the  agglutination  reaction  to  the  practical  diagnosis  of 
typhoid  fever,  finding  that  the  serum  of  patients  afflicted  with  this 
disease  showed  agglutinating  power  over  the  typhoid  bacillus  at 
early  stages  in  the  course  of  the  fever.  The  reaction,  thus  practically 
applied  to  clinical  diagnosis,  was  soon  shown  to  be  of  great  im- 
portance in  its  bearing  on  bacteriological  species  differentiation. 
Since  animals  immunized  against  a  definite  species  of  bacteria  ac- 
quire in  their  sera  specific  agglutinating  powers  for  these  bacteria 
and  at  best  only  slight  agglutinating  powers  for  other  species,  im- 
mune sera  can  be  used  extensively  in  differentiating  between  bac- 
terial varieties. 

Agglutination  may  be  observed  microscopically  or  macroscopic- 
ally.  Bacteria  brought  into  contact  with  agglutinating  serum  in 
the  hanging  drop  rapidly  lose  their  motility,  if  motile,  as  in  the 
case  of  typhoid  bacilli,  and  gather  together  in  small  clumps  or 
masses.  The  microscopic  picture  is  striking  and  easily  recognized 
and  the  reaction  takes  place  with  varying  speed  and  completeness, 
according  to  the  strength  of  the  agglutinating  scrum. 


11  Metchnikoff,  "Etudes  sur  I'lmmunite,"  IV  Memoir,  1891. 

12  Charrin  et  Eager,  Compt.  rend,  de  la  soc.  de  biol.,  1889. 

13  Gruber  und  Durham,  Munch,  med.  Woch.,  1896. 


SENSITIZING  ANTIBODIES  283 

As  the  reaction  approaches  completeness,  the  clumps  grow  larger, 
individual  microorganisms  become  more  and  more  scarce,  finally 
leaving  the  medium  between  clumps  entirely  clear.  While  the  clump- 
ing of  a  motile  organism  suggests  that  motility  has  something  to 
do  with  the  coming  together  in  clumps,  it  nevertheless  has  no  rela- 
tion whatever  to  agglutination,  motile  and  non-motile  organisms 
alike  being  subject  to  the  reaction. 

Macroscopically  observed,  in  small  test  tubes  or  capillary  tubes, 
agglutination  evidences  itself  by  the  formation  of  flake-like  masses 
which  settle  into  irregular  heaps  at  the  bottom,  leaving  the  super- 
natant fluid  clear,  in  distinct  contrast  to  the  even  flat  sediment  and 
the  clouded  supernatant  fluid  of  the  control.  Macroscopically,  too, 


FIG.  34. — MICROSCOPIC  AGGLUTINATION  REACTION. 

agglutination  is  evidenced  when  bacteria  are  grown  in  broth  to 
which  immune  serum  has  been  added.  Instead  of  evenly  clouding 
the  broth,  the  microorganisms  develop  in  clumps  or  chains. 

Another  phenomenon  probably  produced  by  agglutinins  is  the 
so-called  ''thread-reaction"  of  Pfaundler.14  This  consists  in  the 
formation  of  long  convoluted  threads  of  bacterial  growth  in  the 
hanging  drop  of  dilute  immune  serum  after  twenty-four  hours.  Very 
strict  specificity  is  attributed  to  this  reaction  by  Pfaundler. 

Agglutinins  act  upon  dead  as  well  as  upon  living  bacteria.  For 
the  microscopic  tests  bacterial  emulsions  killed  by  formalin  were 
introduced  by  Neisser. 

Ficker15  has  recently  succeeded  in  preparing  an  emulsion   of 


14  Pfaundler,  Cent,  f .  Bakt.,  xxiii,  1898. 

15  Fic'ker,  Berl.  klin.  Woch.,  1903. 


284 


INFECTION  AND   IMMUNITY 


typhoid  bacilli,  which  is  permanent  and  may  be  kept  indefinitely, 
and  may  be  employed  for  macroscopic  agglutinations.16 

Attention  has  been  called  by  various  workers  to  a  source  of 
error  in  all  these  methods,  known  as  pseudo-clumping.17  The  causes 
for  such  clumping  not  due  to  agglutinins  seem  to  lie  in  the  presence 
of  blood  cells  in  the  serum  or  excessive  acidity  of  the  culture 


FIG.  35. — MACROSCOPIC  AGGLUTINATION.  Dilutions  from  1  to  10  in  to  1  to  1,000. 
The  first  tube  contains  a  1  :  20  control  with  the  bacteria  and  normal  serum 
Agglutination  complete  in  the  tubes  marked  10,  20,  50,  100,  r 

medium.18  In  fact,  agglutination  of  bacteria  by  acids  in  definite 
concentration  can  be  Carried  out  and  seems  to  depend  directly  upon 
the  hydrogen  ion  concentration. 

While  the  microscopic  methods  are  more  suitable  for  clinical- 


16  Exact  method  of  -production  of  ' '  Ficker  's  Diagnosticum "  is  a  proprietary 
secret. 

37  Savage,  Jour,  of  Path,  and  Bact.,  1901. 

18  Biggs  and  Park,  Amer.  Jour,  of  Med.  Sci.,  1897;  Block,  Brit.  Med.  Jour., 
1897. 


SENSITIZING  ANTIBODIES  285 

diagnostic  purposes,  because  of  the  smaller  amounts  of  blood  re- 
quired, the  macroscopic  tests  are  far  preferable  for  the  purposes 
of  bacterial  differentiation  and  research.  Greater  exactitude  of 
dilution  is  possible  when  dealing  with  larger  quantities ;  microscopic 
unevenness  in  the  bacterial  emulsion  does  not  become  a  source  of 
error ;  and  positive  and  negative  reactions  are  more  sharply  defined. 
Nature  of  Agglutinins. — Gruber  and  Durham,19  the  discoverers 
of  agglutinins,  at  first  advanced  the  opinion  that  the  agglutinins 
were  identical  with  the  immune  body  concerned  in  the  Pfeiffer 
reaction,  which  by  injuring  the  bacteria  rendered  them  susceptible 
to  the  alexins.  Pfeiffer20  and  Kolle21  claimed,  however,  that  by  the 
addition  of  cholera  vibrio  to  immune  serum,  the  agglutinins  could 
be  completely  absorbed,  or  used  up,  while  bacteriolytic  substances 
still  remained.  The  same  authors  demonstrated  that  immune  serum, 
preserved  for  several  months,  would  lose  its  agglutinins  without  a 
corresponding  loss  of  bacteriolytic  power.  It  has  also  been  shown 
since  then,  by  these  and  other  authors,  that  the  agglutinins  and 
the  bactericidal  substances  are  in  no  way  parallel  in  their  develop- 
ment, and  that  strongly  agglutinating  sera  may  be  extremely  weak 
in  bactericidal  substances  and  vice  versa.  We  ourselves  are  not  at 
all  sure  that  this  proves  sufficiently  that  agglutinins  and  bacteriolysins 
are  distinct  substances.  There  are  many  reasons  to  believe  that 
it  requires  a  considerably  more  powerful  sensitization  to  produce 
agglutination  than  it  does  to  make  an  antigen  amenable  to  alexin 
action,  or  the  action  of  leucocytes,  and  also  the  actual  agglutination 
or  clumping  depends  upon  environmental  conditions  in  which  vis- 
cosity of  the  menstruum,  the  presence  of  electrolytes,  and  perhaps 
also  the  condition  of  the  antibody  in  the  serum,  namely,  whether 
or  not  it  is  relatively  free  or  is  bound  up  with  serum  proteins,  play 
a  part.  Bordet  has  shown  very  definitely  that  clumping  does  not 
take  place  without  the  presence  of  electrolytes.  If  bacteria  are 
sensitized  heavily,  or,  in  other  words,  allowed  to  absorb  antibody, 
and  then  washed  and  resuspended  in  distilled  water,  they  do  not 
agglutinate.  A  small  amount  of  salt  solution  added  to  such  a 
mixture  brings  about  rapid  agglutination.  In  this,  as  well  as  in 
the  phenomena  of  acid  agglutination,  the  clumping  of  the  bacteria 


19  LOG.  cit. 

20  Pfeiffer,  Dent.  med.  Woch.,  1896. 

21  Pfeiffer  und  Kolle,  Cent,  f .  Bakt.,  xx,  1896. 


28(3  INFECTION   AND   IMMUNITY 

is  entirely  analogous  to  the  clumping  of  colloidal  suspensions  of 
any  kind,  and  if  we  consider  that  bacterial  suspensions  are  in  many 
other  respects  similar  to  colloidal  suspensions,  namely,  in  their 
negative  charge  and  their  wandering  to  the  positive  pole  in  neutral 
solutions,  in  kataphoresis  experiments,  it  becomes  apparent  that  the 
actual  clumping  or  agglutination  is  a  purely  physical  phenomenon, 
determined  by  the  colloidal  equilibrium  of  the  bacteria  in  suspension. 

Altogether,  we  ourselves  are  inclined  to  regard  the  so-called 
agglutinins  as  identical  with  other  sensitizers,  and  the  actual  clump- 
ing as  a  colloidal  precipitation  of  organisms  that  have  been  altered 
in  their  suspension  equilibrium  as  a  result  of  the  union  with  the 
antibodies  together  with  some  serum  protein. 

Agglutinated  bacteria22  are  not  killed  by  the  act  of  agglutination 
and  are  often  as  virulent  as  non-agglutinated  cultures.  Metchnikoff 
assigned  to  them  a  secondary  role  in  relation  to  protection,  but 
it  is  quite  likely  from  more  recent  observations,  that  they  do  par- 
ticipate distinctly  in  the  protective  mechanism.  Recent  work  by 
Bull  seems  to  indicate  that  bacteria  are  agglutinated  in  vivo  as  a 
sort  of  preliminary  step  to  phagocytosis. 

The  agglutinins,  furthermore,  unlike  the  bactericidal  substances 
in  sera,  remain  active  after  exposure  to  temperatures  of  over  55°  C., 
some  of  them  withstanding  even  65°  to  70°,  and  can  not  be  reac- 
tivated by  the  subsequent  addition  of  normal  serum.  These  facts 
definitely  preclude  the  participation  in  the  reaction  of  the  alexin 
or  complement. 

Production  of  Agglutinins. — Just  as  normal  sera  contain  small 
quantities  of  bactericidal  substances,  so  do  they  contain  agglutinins 
in  small  amount.  In  a  general  way  these  "normal  agglutinins" 
have  the  same  nature  as  the  immune  agglutinins,  and  their  presence 
is  probably  traceable  to  the  various  microorganisms  parasitic  upon 
the  human  and  animal  body. 

As  a  matter  of  fact,  the  blood  serum  of  new-born  guinea-pigs 
hardly  ever  contains  agglutinin  for  B.  coli,  while  that  of  adults 
acts  upon  these  bacilli  in  dilutions  of  1 :20.23  Similarly,  infants 
show  lower  normal  agglutinating  values  than  adults.24 

Agglutinins  may  be  produced  in  the  sera  of  animals  by  the  intro- 


22  Mesnil,  Ann.  de  1'inst.  Pasteur,  1898. 

2S  Kraus  und  Low,  Gesell.  d.  Aerzte,  Wien,  1899. 

"Pfaundler,  Jahrb.  f.  Kinderheilk,.,  Bd.-50. 


SENSITIZING  ANTIBODIES  287 

duction  of  microorganisms  subcutaneously,  intravenously,  or  intra- 
peritoneally.  The  intravenous  method  seems  to  give  the  most 
abundant  and  speedy  results.25  The  formation  of  agglutinins  is  a 
reaction  to  the  body  substances  of  the  bacteria  themselves,  rather  than 
to  their  toxic  products.  Thus  agglutinins  are  produced  in  response 
to  the  introduction  of  dead  bacteria  and  soluble  extracts  of  cultures. 
Pathogenicity26  does  not  influence  agglutinin  formation  to  any  great 
extent,  non-pathogenic  as  well  as  pathogenic  -giving  rise  to  these 
substances  in  serum.  As  a  rule,  however,  agglutinins  are  more 
easily  produced  against  a  virulent  than  against  fully  virulent  strains 
of  bacteria  of  the  same  species. 

Agglutinins  can  be  produced  with  all  the  known  bacteria,  but 
great  difficulty  may  be  experienced  in  producing  them  with  cap- 
sulated  organisms  such  as  the  pneumococcus  mucosum  and  the  Fried- 
lander  bacillus,  since  the  capsule  seems  to  insulate  such  bacteria 
against  reactions  witli  serum.  It  is  possible  to  agglutinate  such 
capsulated  bacteria  often  only  by  the  method  of  Forges,  the  pre- 
liminary destruction  of  the  capsule  with  weak  acid  and  heat.  As 
a  rule,  the  agglutinins  appear  in  the  blood  of  animals  three  to  six 
days  after  the  introduction  of  bacteria.  From  the  third  to  the  sixth 
day  they  rapidly  increase  to  a  maximum  at  the  seventh  to  thirteenth 
day.  They  then  fall  off  rapidly  until  they  reach  a  level  at  which 
they  remain  for  a  long  period  without  very  considerable  change. 
Curves  to  illustrate  these  phases  have  been  constructed  by  Jorgensen 
and  Madsen.27 

The  Reaction  between  Agglutinin  and  Agglutinin-Stimulating 
Substances  (Agglutinogen) . — The  fact  that  agglutinin  can  be  removed 
from,  or  absorbed  out  of,  serum  by  the  specific  bacilli  which  have  led 
to  its  formation  indicates  that  there  is  in  the  act  of  agglutination 
a  combination  between  the  agglutinin  and  the  agglutinin-stimulating 
substance  (agglutinogen).  It  is  likely  that  this  combination  is  of 
a  chemical  nature,  since,  as  we  have  mentioned,  agglutinins  result 
from  the  injection  of  bacterial  extracts  as  well  as  from  the  intro- 
duction of  living  bacteria.  The  probability  that  the  process  follows 
chemical  laws  of  combination  is  furthermore  strengthened  by  the 
work  of  Joos28  and  others,  who  have  demonstrated  that  definite 

23  Hoffmann,  Hyg.    Eundschau,  1903. 
"Nicolle,  Ann.  de  1'inst.  Pasteur,  1898. 

27  Jorgensen  and  Madsen,  Festschrift,  Kopenhagen,  1902. 

28  Joos,  Zeits.  f.  Hyg.,  xxxvi,  1901. 


288  INFECTION  AND   IMMUNITY 

quantitative  relations  exist  between  the  agglutinin-stimulating  sub- 
stances and  the  agglutinins.  Every  agglutination  reaction,  there- 
fore, will  vary  in  its  degree  of  completeness  with  the  quantities  of 
agglutinin  and  agglutinogeii,  a  fact  which  makes  it  necessary,  es- 
pecially for  clinical  tests,  to  preserve  a  certain  uniformity  in  the 
quantity  and  density  of  the  bacterial  culture  or  emulsion  employed. 

SPECIFICITY. — From  the  very  beginning,  Gruber  and  Durham29 
had  claimed  specificity  for  the  agglutination  reaction,  and  in  this 
sense  it  was  clinically  utilized  by  Widal  for  the  diagnosis  of  typhoid 
fever.  It  was  noticed,  however,  even  by  these  earliest  workers, 
that  the  serum  of  an  animal  immunized  against  one  microorganism 
would  often  agglutinate,  to  a  less  potent  degree,  other  closely  re- 
lated species.  Thus,  the  serum  of  a  typhoid-immune  animal  may 
agglutinate  the  typhoid  bacillus  in  dilutions  of  1 :1,000,  and  the 
colon  bacillus  in  dilutions  as  high  as  1:200;  while  the  agglutinating 
power  of  normal  serum  for  the  colon  bacillus  rarely  exceeds  1 :20. 
The  specificity  of  the  reaction  for  practical  purposes,  thus,  is  not 
destroyed  if  proper  dilutions  is  carried  out,  the  degree  of  agglutinin 
formation  being  always  far  higher  for  the  specific  organism  used 
in  immunization  than  it  is  for  allied  organisms.  The  specific  immune- 
agglutinin  in  such  experiments  is  spoken  of  as  the  chief  agglutinin 
(hauptagglutinrn),  and  the  agglutinins  formed  parallel  with  it,  as 
the  partial  agglutinin  (metagglutinin),  terms  introduced  by  Was- 
sermann.  Hiss  has  spoken  of  these  as  major  and  minor  agglutinins. 
The  relative  quantities  of  the  specific  chief  agglutinin  and  partial 
agglutinins  present  in  any  immune  serum  depend  upon  the  individual 
cultures  used  for  immunization,  and  the  phenomenon  is  probably 
dependent  upon  the  fact  that  certain  elements  in  the  complicated 
bacterial  cell-body  may  be  common  to  several  species  and  find  com- 
mon receptors  in  the  animal  body.  Whenever  an  immune  serum 
agglutinates  a  number  of  members  of  the  group  related  to  the 
specific  organism  used  for  its  production,  the  reaction  is  spoken  of 
"  group  agglutination. ' ' 

The  partial  agglutinins  (met agglutinins)  have  been  extensively 
studied  by  Castellani30  and  others,  by  a  method  spoken  of  as  the 
" absorption  method."  This  consists  in  the  separate  addition  of 
bacterial  emulsions  (agglutinogens)  of  the  various  species  concerned 


29  Gruber  und  Durham,  loc.  cit.  r 

80  Castellani,  Zeits.  f.  Hyg.,  xl,  1902. 


SENSITIZING  ANTIBODIES  289 

in  a  group  agglutination,  to  the  agglutinating  serum.  In  this  way, 
specific  and  partial  agglutinins  can  be  separately  removed  from  the 
immune  serum  by  absorption — each  by  its  corresponding  agglutino- 
gen.  In  such  experiments  all  agglutinins  will  be  removed  by  the 
organisms  used  for  immunization,  a  partial  removal  only  resulting 
from  the  addition  of  allied  strains.  This  method  has  thrown  much 
light  upon  the  intimate  relations  existing  between  members  of  vari- 
ous' bacterial  species,  and  has  been  particularly  valuable  in  the  study 
of  the  typhoid-colon-dysentery  group.  It  is  important  to  mention, 
however,  that  "groups"  as  determined  by  agglutination  tests  do 
not  always  correspond  to  classifications  depending  upon  mor- 
phological and  cultural  characteristics. 

An  interesting  phenomenon  of  great  practical  importance,  which 
has  been  noticed  by  a  number  of  observers,  and  which  may  often 
be  encountered  in  routine  agglutination  tests,  is  the  frequent  failure 
of  a  strongly  agglutinating  serum  to  produce  agglutination  if  used 
in  concentration,  while  in  dilutions  it  produces  a  characteristic  re- 
action. This  has  been  explained  theoretically  by  what  is  known 
as  the  "proagglutinoid  zone."  It  is  assumed  that  agglutinins  may 
deteriorate  as  do  toxins  and  be  converted  into  substances  which  are 
capable  of  combining  with  agglutinogen  without  causing  agglutina- 
tion. Such  substances,  as  we  will  see  in  discussing  Ehrlich's  views 
on  the  structure  of  agglutinins,  may  have  a  stronger  affinity  for 
agglutinogen  than  the  agglutinins  themselves,  and  are,  therefore, 
termed  "proagglutlnoids."  In  strongly  agglutinating  sera  these 
proagglutinoids  may  be  present  in  considerable  quantities  and  pre- 
vent the  combination  of  agglutinin  with  agglutinogen.  In  dilution, 
this  proagglutinoid  action  would  naturally  become  weaker  and  of 
no  actual  significance  in  obscuring  the  reaction. 

Agglutination,  like  other  immune  phenomena,  is  a  manifestation 
of  broad  biological  laws  and  not  limited  to  bacteria.  Thus,  as 
hemolysins  are  produced  by  the  injection  of  red  blood  cells,  so 
hem  agglutinins,  or  substances  which  clump  together  red  blood  cells, 
are  similarly  formed. 

The  theoretical  considerations  concerning  the  nature  of  agglu- 
tinins are  discussed  below,  together  with  a  similar  section  on  the 
precipitins. 

Precipitation. — R.  Kraus,31  of  Vienna,  demonstrated  that  the  sera 
of  animals  immunized  aaginst  B.  pestis,  B.  typhosus,  and  Vibrio 

31  Kraus,  Wien.  klin.  Woch.,  1897, 


290  INFECTION   AND   IMMUNITY 

cholerae,  when  mixed  with  the  clear  filtrate  of  bouillon  cultures  of 
the  respective  organisms,  produce  microscopically  visible  precipi- 
tates. These  precipitates  occurred  only  when  filtrate  and  immune 
serum  were  homologous,  i.  e.,  when  the  animal  from  which  the  serum 
had  been  obtained  had  been  immunized  by  the  same  species  of 
microorganism  as  that  which  was  used  in  the  test ;  it  was  for  this 
reason  Kraus  spoke  of  them  as  "specific  precipitates."  It  was 
evident,  therefore,  that  during  the  process  of  active  immunization 
with  these  organisms,  a  specific  antibody  had  been  produced  in  the 
serum  of  the  treated  animal,  which,  because  of  its  precipitating 
quality,  was  named  "precipitin. "  This  peculiar  reaction  was  soon 
found  to  hold  good,  not  only  for  the  bacteria  used  by  Kraus,  but 
also  for  other  bacteria,  few  failing  to  stimulate  the  production  of 
specific  precipitins  in  the  sera  of  immunized  animals.  The  phenom- 
enon of  precipitation,  however,  is  not  limited  to  bacterial  immuniza- 
tion, but  has  been  found,  like  the  phenomena  of  agglutination  and 
lysis,  to  depend  upon  biological  laws  of  broad  application.  Thus, 
Bordet32  found  that  the  blood  serum  of  rabbits  treated  with  the 
serum  of  the  chicken  gave  a  specific  precipitate  when  mixed  with 
chicken  serum.  Tchistovitch33  demonstrated  a  similar  reaction  with 
the  sera  of  rabbits  treated  with  horse  and  eel  sera.  By  the  injection 
of  milk,  Wassermann,34  Schutze,35  and  others  produced  an  antibody 
which  precipitated  the  casein  of  the  particular  variety  of  milk 
employed  for  immunization.  The  reaction  was  thus  applicable  to 
many  albuminous  substances.  These  substances,  because  of  their 
precipitin-stimulating  quality,  are  called  "preeipitinogens." 

Nature  of  Precipitins. — The  precipitins,  like  the  agglutinins,  may 
be  inactivated  by  heating  to  from  60°  to  70°  C.,  and  can  not  be 
reactivated  by  the  addition  of  normal  serum  or  by  any  other  known 
method.  Such  inactivated  precipitin,  however,  while  unable  to 
produce  precipitates,  has  not  lost  its  power  of  binding  the  precipi- 
tinogen.  This  is  shown  by  the  fact  that  the  inactivated  precipitin, 
when  mixed  with  precipitinogen,  will  prevent  subsequently  added 
fresh  precipitin  from  causing  a  reaction.  From  these  facts  the  con- 
clusion has  been  drawn  that  precipitin,  like  toxin,  is  built  up  of 


32  Bordet,  Ann.  de  Pinst.  Pasteur,  1899. 

83  Tchistovitch,  Ann.  de  1'inst.  Pasteur,  1899. 

84  Wassermann,  Deut.  med.  Woch.,  29,  1900. 
KSchiitse,  Zeit.  f.  Hyg.,  1901. 


SENSITIZING  ANTIBODIES  291 

two  atom  groups,36  a  stable  haptophore  and  a  labile  precipitophore 
group.  By  the  destruction  of  the  latter,  an  inactive,  yet  neutralizing 
substance  is  produced  which  is  spoken  of  as  "precipitoid."  The 
precipitoids,  like  protoxoids,  have  a  higher  affinity  for  precipitinogen 
than  the  unchanged  precipitin,  and  thus  are  able  to  prevent  the 
action  of  these. 

Our  own  opinion  would  rather  incline  toward  regarding  the 
precipitins  as  identical  in  structure  with  sensitizer  or  amboceptor — 
being  in  fact  * '  albuminolysins "  in  the  sense  of  Gengou.  This  prob- 
lem is  too  complex  to  be  discussed  in  detail  in  a  summary  of  im- 
munity as  brief  as  the  one  here  presented. 

Specificity. — The  specificity  of  precipitins  is  a  question  of  the 
greatest  importance,  since,  as  we  shall  see,  these  bodies  have  been 
used  extensively  for  the  differentiation  of  animal  proteins.  In  re- 
gard to  bacterial  precipitins  it  may  be  said  that,  just  as  in  agglu- 
tination, there  is  in  precipitation,  a  certain  degree  of  "group  reac- 
tion." The  precipitin  obtained  with  a  colon  bacillus,  for  instance, 
will  cause  precipitation  with  culture-filtrates  of  closely  allied  or- 
ganisms, though  in  a  less  marked  degree.  According  to  Kraus,  such 
confusion  may  be  easily  overcome  by  the  proper  use  of  dilution  and 
quantitative  adjustment,  similar  to  that  used  in  agglutination  tests. 
Norris37  found  that  the  precipitates  given  by  immune  sera  with  the 
filtrates  of  the  homologous  bacteria  were  invariably  heavier  than 
those  given  with  allied  strains  and  that  the  latter  could  be  eliminated 
entirely  by  sufficient  dilution. 

Specificity  becomes  of  still  greater  importance  in  the  forensic  use 
of  the  precipitin  reaction  introduced  by  Uhlenhuth,38  Wassermann 
and  Schiitze,39  and  Stern.40  These  authors  found  that  the  precipitin 
reaction  furnished  a  means  of  distinguishing  the  blood  of  one  species 
from  that  of  another.  Thus,  blood  spots,  dissolved  out  in  normal 
salt  solution,  could  be  recognized  by  this  reaction  as  originating 
from  man  or  from  an  animal,  even  after  months  of  drying  and  in 
dilutions  as  high  as  1 :50,000.  Since  the  value  of  this  test  depends 
entirely  upon  the  strict  specificity  of  the  reaction,  this  question  has 


u  Kraus  und  v.  Pirquet,  Cent.  f.  Bakt.,  Orig.  Bd.  xxxii. 

37  Norris,  Jour.  Inf.  Dis.,  i,  3,  1904. 

38  Uhlenhuth,  Deut.  med.  Woch.,  xlvi,  1900;  vi  and  xvii,  1901. 

39  Wassermann  und-  Schutze,  Berl.  klin.  Woch.,  vi,  1901. 

40  Stern,  Deut.  med.  Woch.,  1901. 


292  INFECTION  AND   IMMUNITY 

been  studied  with  especial  care,  notably  by  Nuttall.41  All  who  have 
investigated  the  subject  find  the  only  important  source  of  confusion 
in  the  blood  of  the  anthropoid  apes.  The  specificity  of  the  reaction, 
too,  has  been  found  to  depend  very  closely  upon  the  amount  of 
precipitin  in  the  serum  employed.  If  a  highly  immune  serum  is 
insufficiently  diluted,  the  reaction  loses  much  of  its  specific  value.42 
This  source  of  error  is  easily  eliminated  in  practice  by  careful  con- 
trol and  tit-ration  of  the  sera  used  for  the  tests. 

Unlike  agglutinins,  precipitins  have,  so  far,  not  been  demon- 
strated in  normal  sera.43 

Theoretical  Considerations  Concerning  Agglutinins  and  Precipi- 
tins.— We  have  seen  that  Ehrlich  evolved  his  theories  of  antibody 
formation  from  his  early  views  upon  the  absorption  of  nutritive 


Cell  -used  for  i 


FIG.  36.  —  EHRLICH'S  CONCEPTION  OF  THE  STRUCTURE  OP  AGGLUTININS  AND 

PRECIPITINS. 

substances  by  the  body  cells,  and  we  have  followed,  in  more  or 
less  detail,  the  steps  of  his  reasoning  as  he  developed  his  hypothesis 
in  its  application  to  the  antitoxic  and  the  lytic  substances.  There 
still  remained  the  agglutinins  and  precipitins,  bodies  which  because 
of  their  individual  characteristics  can  be  classed  neither  with  the 
group  of  antitoxic,  nor  with  that  of  the  lytic  substances.  These 
two  antibodies,  while  by  no  means  identical,  possess  the  common 
characteristics  of  being  more  thermostable  than  the  bacteriolytic 
substances,  and  of  being  insusceptible  to  reactivation  by  normal 
serum.  It  is  plain,  therefore,  that  both  agglutinating  and  precipi- 
tating reactions  take  place  without  the  co-operation  of  complement. 
The  substances  which  give  rise  to  precipitins  and  agglutinins,  more- 


41  Nuttall,  Brit.  Med.  Jour.,  i,  1901;  ii,  1902. 
4-Kister  und  Wolff,  Zeit.  f.  Medizinal-Beamte,  1902, 
43  Kraus,  loe.  cit.,  and  Norris,  loc.  cit. 


SENSITIZING  ANTIBODIES  203 

over,  are  not  of  the  relatively  simple  soluble  character  of  the  toxins, 
but  are  intrinsic  portions  of  complex  albuminous  molecules,  com- 
parable to  and  often  identical  with  the  true  nutritive  substances. 
For  these  reasons  Ehrlich  believes  that  the  cell-receptors  for  the 
various  substances  which  give  rise  to  agglutinins  and  precipitins 
are  neither  of  the  simple  structure  of  the  toxin  receptor,  nor  of  the 
double-haptophore  nature  of  the  bacteriolytic  receptors,  but  contain 
a  single  haptophore  group  for  the  anchorage  of  the  ingested  material 
and  at  the  same  time  a  constantly  attached  zymophore  group  or 
ferment  by  means  of  which  the  anchored  substance  is  transformed 
preparatory  to  its  absorption  by  the  cell  protoplasm.  For  the  sake 
of  clearness,  this  form  of  receptor  may  be  compared  to  a  bacteriolytic 
or  hemolytic  amboceptor  with  a  permanently  attached  and  insepa- 
rable complement. 

Three  forms  of  receptors,  then,  are  proposed  by  Ehrlich  in  ex- 
planation of  all  known  varieties  of  antibodies.  The  first,  the  sim- 
plest side  chains  of  the  body  cells,  he  calls  "receptors  or  haptines 
of  the  first  order. ' '  These,  overproduced  and  cast  off,  constitute  the 
antitoxin  and  antiferments.  Next  "haptines  of  the  second  order" 
are  the  receptors  planned  both  for  the  anchorage  and  further  diges- 
tion of  antigens.  These,  free  in  the  circulation,  are  the  precipitins 
and  agglutinins.  "Haptines  or  receptors  of  the  third  order"  are 
merely  able  to  anchor  a  suitable  substance,  but  exert  no  further 
action  upon  it  until  re-enforced  by  the  complement  normally  present 
in  the  serum.  These,  free  in  the  circulation,  with  a  chemical  group 
having  avidity  for  the  antigen,  and  another  complementophile  group, 
are  the  amboceptors  or  immune  bodies  of  bacteriolytic,  cytolytic, 
and  hemolytic  sera. 

We  cannot,  in  a  general  work  of  this  kind,  go  into  a  detailed 
discussion  of  the  many  complex  problems  involved  in  regard  to 
the  structure  of  antibodies.  Again  we  will  refer  the  reader  to  the 
larger  works  on  this  subject.  It  is  our  opinion,  however,  that  the 
above  views  of  Ehrlich  can  no  longer  be  maintained  in  the  light 
of  present  knowledge. 

Bordet,44  points  out  that  the  conception  of  Ehrlich  rests  upon 
the  basis  of  a  number  of  undemonstrated  hypotheses.  He  asserts, 
and  with  justice,  that  it  has  never  been  shown  beyond  question  that 


44  Bordet,  Kesume  of  Immunity  in  Bordet 's  ' i  Studies  in  Immunity, ' '  transl.  by 
Gay,  Wiley  &  Sons,  1909, 


294  INFECTION   AND   IMMUNITY 

the  antibodies,  free  in  the  serum,  are  identical  with  the  receptors 
of  the  body  cells  upon  which  the  antigen  originally  acts. 

In  regard  to  agglutinins,  Ehrlich,  as  we  have  seen,  believes  that 
it  is  the  agglutinin  itself  which,  first  uniting  with  its  antigen  by 
its  haptophore  group,  then  causes  clumping  by  its  zymophore  group. 
Now,  as  a  matter  of  fact,  Bordet45  has  shown  that  it  is  not  the 
agglutinin  itself  which  agglutinates,  but  that  agglutinin  with  its 
antigen  forms  a  complex  which  is  then  agglutinable  by  the  salt 
present  in  the  solution.  This  conclusion  seems  borne  out  by  the 
later  work  of  Gengou,46  Landsteiner  and  Jagic,47  and  others,  who 
have  shown  that  bacteria  which  have  absorbed  other  substances, 
such  as  uranium  compounds,  colloidal  silicic  acid,  etc.,  are  subse- 
quently agglutinable  by  salts.  In  consequence,  from  these  and  other 
observations,  Bordet  concludes  that  it  is  neither  necessary  nor  ac- 
curate for  the  explanation  of  these  phenomena,  to  assume  the  con- 
ditions conceived  by  Ehrlich,  but  that  the  phenomenon  of  agglutina- 
tion consists  primarily  of  the  union  of  the  antibody  with  its  antigen 
in  a  colloidal  suspension,  and  that  the  actual  subsequent  agglutina- 
tion is  a  purely  secondary  phenomenon  which  depends  possibly  upon 
a  change  in  the  physical  properties  of  the  emulsion — upon  "its  col- 
loidal stability."  A  similar  condition  he  assumes  for  precipitins. 

Without  being  able  in  the  limited  space  available  to  go  into  a 
detailed  discussion  of  the  large  volume  of  work  which  has  appeared 
on  this  subject,  we  may  say  that  it  is  our  opinion  at  present  that 
the  evidence  largely  points  in  the  direction  indicated  by  Bordet, 
namely,  that  the  essential  feature  of  all  these  reactions  is  the  specific 
union  of  an  antigen  with  its  antibody,  that  thereby  the  physical 
or  chemical  condition  of  the  antigen  is  so  changed  that  it  now 
becomes  less  stable  and  is  agglutinated  or  precipitated  by  such 
physical  influences  as,  for  instance,  the  presence  of  an  electrolyte. 
The  work  of  Neisser  and  Friedmann48  has  shown  that  bacteria  that 
have  absorbed  agglutinin  are  agglutinated  by  concentrations  of  salt 
far  less  than  is  necessary  to  agglutinate  or  precipitate  the  normal 
bacteria. 

Our  own  opinion,  set  forth  in  a  number  of  experimental  studies, 
would  go  even  further  than  this.  We  incline  to  the  belief  that  all 

4r>  Bordet,  Ann.  de  Pinst.  Pasteur,  1899. 

46  Gengou,  Annal.  Past.,  1904. 

47  Landsteiner  mid  Jagic,  Wien.  klin.  Woch.,  iii,  1904. 

48  Neisser  and  Friedmann,  Munch,  med.  Woch.,  1904,  li.  465-827. 


SENSITIZING  ANTIBODIES  295 

antibodies,  including  the  so-called  amboceptors  or  sensitizers  that 
take  part  in  the  phenomena  of  lysis  and  bactericidal  action  are 
essentially  of  one  type;  that  the  fundamental  phenomenon  is  the 
union  of  the  antigen  with  the  specific  antibody  or  its  "sensitiza- 
tion;"  that  by  such  sensitizatioii  the  antigen  is  now  rendered  on 
the  one  hand  more  easily  agglutinable  or  precipitable,  on  the  other 
may  be  rendered  more  amenable  to  the  action  of  the  alexin  or  com- 
plement or  to  phagocytosis.  The  agglutination  and  precipitation 
phenomena,  moreover,  are  merely  evidences  of  the  fact  that  these 
substances  are  in  colloidal  suspension  and  are  influenced  by  agencies 
which  produce  precipitations  in  such  suspension.  It  is  interesting 
to  note  in  this  connection,  also,  that  bacteria  in  neutral  suspension 
carry  negative  charges  which  can  be  weakened  by  sensitization  with 
serum  and  weakened  or  reversed  by  the  addition  of  acid.  These 
points  tend  to  strengthen  such  a  point  of  view. 

The  degree  of  acidity  necessary  to  reverse  the  normal  negative 
charge  of  bacteria  corresponds  roughly  to  that  at  which  growth  is 
inhibited.  This  has  led  us  to  speculate  whether  or  not  vitality  of 
bacteria  and  the  negative  charge  may  be  related. 

Facts  Concerning  Alexin  or  Complement. — Muir  and  Browning 
claim  that,  on  the  filtration  of  serum,  amboceptor  or  immune  body 
will  pass  through  the  filter,  whereas  alexin  or  complement  is  held 
back. 

This  retention  of  complement  by  filters  occurs  only  when  new 
filters  are  used,  and  this  is  probably  due  to  adsorption  or  comple- 
ment by  the  finely  divided  substances  which  make  up  the  filter  and 
not  due  to  retention  because  of  the  large  size  of  the  complement 
molecule. 

Alexin  can  be  inactivated  by  shaking  as  well  as  by  heat  when 
diluted  1 :10  and  shaken  for  about  20  minutes  in  salt  solution.  Ac- 
cording to  Gramenitski  it  is  spontaneously  partially  reactiviated 
on  standing. 

Alexin  is  dependent  upon  the  total  volume  of  the  mixture  in 
which  it  acts,  i.e.,  upon  concentration,  the  same  actual  quantity  of 
complement  acting  more  strongly  in  higher  than  in  lower  concen- 
trations, this  not  being  true  of  amboceptor  or  sensitizer  which  acts 
in  direct  proportion  to  its  actual  quantity  independent  of  the  con- 
centration. 

Alexin  is  inhibited  by  hypertonic  salt  solution  and  can  be  pre- 
served in  15-25  per  cent  salt  concentration  for  weeks  in  the  icebox, 


296  INFECTION   AND   IMMUNITY 

resuming  its  activity  when  diluted  to  isotonicity  with  distilled  water. 
Removal  of  salt  by  dialysis  or  other  means  of  globulin  precipitation 
divides  the  complement  into  two  fractions,  the  globulin  fraction  and 
the  albumin  fraction,  neither  of  which  will  act  alone,  but  which 
together  possess  the  properties  of  undivided  complement.  The 
globulin  fraction  attaches  directly  to  the  sensitized  cells  and  is  there- 
fore spoken  of  by  German  investigators  as  "mid-piece."  The  al- 
bumin fraction  acts  upon  the  sensitized  cells  only  after  attachment 
of  the  globulin  fraction  and  is  therefore  spoken  of  as  "end-piece." 
It  is  seen,  therefore,  that  a  great  many  of  the  properties  of  alexin 
make  it  seem  rather  likely  that  this  substance  is  quite  similar  to 
ferments  in  its  action. 

The  Fixation  of  Complement  by  Precipitates. — It  has  been  found 
by  Gengou49  and  confirmed  by  Moreschi,  Gay,50  and  others,  that 
when  the  serum  of  an  animal  immunized  with  the  serum  of  another 
species  or  with  a  foreign  albumin  is  mixed  with  a  solution  of  the 
substance  used  in  the  immunization,  the  precipitate  formed  will 
remove  complement  from  the  mixture.  In  other  words,  precipitates 
formed  by  the  reaction  of  precipitin  with  its  antigen  will  fix  com- 
plement. This  is  of  great  importance  in  complement-fixation  tests ; 
for  because  of  insufficient  washing,  the  blood  cells  used  in  producing 
the  hemolytic  amboceptor,  may,  from  the  presence  of  serum,  give 
rise  to  a  precipitin  as  well  as  a  hemolysin.  In  the  test  done  subse- 
quently, a  precipitin  reaction  may  take  place  and  by  thus  removing 
complement  may  give  a  false  result.  The  absorption  of  complement 
by  such  precipitates  takes  place  when  the  two  reacting  factors,  the 
precipitin  and  its  antigen,  are  in  dilution — so  high  a  visible  precipi- 
tate can  not  be  observed.  This  fact,  together  with  others  too  com- 
plicated to  be  discussed  in  this  place,  have  led  us  to  the  belief 
that  the  so-called  precipitins  are  true  sensitizers,  exerting  toward 
unformed  proteins  the  same  function  that  the  so-called  sensitizer  or 
amboceptor  exerts  toward  cellular  formed  antigens.  (See  p.  293.) 

Quantitative  Relationship  Between  Amboceptor  and  Complement. 
— Morgenroth  and  Sachs51  have  succeeded  in  showing  that  within 
certain  limits  an  inverse  relationship  exists  between  these  two  bodies. 
If  for  a  given  quantity  of  red  blood  cells  a  certain  quantity  of 

49  Gengou,  Ann.  Past.,  1902. 
5(1  Gay,  Cent.  f.  Bakt.,  I,  xxix,  1905. 

51  Morgenroth  nnd  Sachs,  ' ( Gesammel.  Arb.  f iir  Immimitatsf orschung. ' '  Berlin, 
Hirschwald,  1904. 


SENSITIZING  ANTIBODIES  207 

amboceptor  and  complement  suffices  to  produce  complete  hemolysis, 
reduction  of  either  the  complement  or  the  amboceptor  necessitates 
an  increase  of  the  other  factor.  As  amboceptor  is  increased,  in  other 
words,  complement  may  be  reduced  and  vice  versa.  This  result  is  of 
great  importance  in  arguing  against  the  original  conception  of  Ehrlich 
in  supposing  these  substance's  to  act  together  unit  for  unit. 

Deviation  of  the  Complement  (Complement-Ablenkung).— It  was 
noticed  by  Neisser  and  Wechsberg52  that  in  mixing  together  bacteria, 
inactivated  bactericidal  immune  serum  (im- 
mune body),  and  complement  in  the  test  tube, 
a  great  excess  of  immune  body  hindered  rather 
than  helped  bactericidal  action.  As  the  amount 
of  immune  body  in  the  mixture  was  carried 
beyond  the  experimental  optimum,  bactericidal 
action  became  less  and  less  pronounced,  and 
was  finally  completed  suspended.  They  explain 
this  by  assuming  that  free  immune  body,  un- 
combined  with  complement,  has  a  greater 
affinity  for  the  bacterial  receptor  than  the  im- 
mune body  combined  with  complement.  The 
complement  is  consequently  diverted  and  pre- 
vented from  activating  the  amboceptor  attached 

,11  .   n        I-,       r*        -i  .  -,.      FIG.  37. — NEISSER  AND 

to  the  bacterial  cell.     Graphically,  the  condi-       WECHSBBRG,S    CoN. 

tions  may  be  illustrated  as  follows :  CEPTION  OF  COMPLE- 

The   above   theory   of   Neisser   and   Wechs-        MENT  DEVIATION. 
berg    is    here    stated    simply    because    of    the 

wide  discussion  it  has  aroused.  In  the  light  of  our  present 
knowledge  concerning  the  relations  between  antigen,  ambo- 
ceptor, and  complement,  their  conception  is  obviously  erroneous. 
The  phenomenon  of  Neisser  and  Wechsberg  is  probably  a  "zone" 
phenomenon,  namely,  an  occurrence  which  depends  upon  the  fact 
that  the  complete  or  incomplete  union  of  colloidal  substances  de- 
pends to  a  very  great  extent  upon  the  relative  concentrations  of 
each,  and  too  high  a  concentration  of  the  anti-serum  in  experiments, 
such  as  those  of  Neisser  and  Wechsberg,  may  result  in  incomplete 
union.  Thus,  it  is  possible  in  many  colloidal  precipitation  phenomena 
to  show  that  too  high  a  concentration  of  one  or  the  other  reacting 
colloid  will  result  in  failure  of  precipitation,  and  in  some  cases,  even 

52  Neisser  und  Wechsberg,  Munch,  med.  Woch.,  xviii,  1901. 


298  INFECTION   AND   IMMUNITY 

when  precipitation  has  taken  place,  dispersion  will  again  occur,  if 
one  or  the  other  component  is  added  in  excess.  These  phenomena 
are  frequently  observed  in  agglutination  and  precipitation  reactions 
where  the  highest  concentrations  of  serum  will  produce  less  pre- 
cipitate, or  perhaps  none  at  all  when  greater  dilutions  produce  heavy 
precipitation. 

Fixation  of  the  Complement. — Bordet  and  Gengou53  in  1901,  de- 
vised an  ingenious  method  of  experimentation  by  which  even  very 
small  quantities  of  any  given  immune  body  (amboceptor)  can  be 
demonstrated  in  serum.  The  term  "fixation  of  complement,"  by 
which  their  method  of  investigation  is  now  generally  known,  ex- 
plains itself,  as  the  steps  of  experimentation  are  followed.  They 
prepared  the  following  mixtures : 

(a)  (b) 

Bacteriolytic  amboceptor  Normal  serum,  heated 

(Plague  immune  serum,  heated) 

+  + 

Plague  emulsion  Plague  emulsion 

+  + 

Complement  Complement 

(Fresh  normal  serum)  (Fresh  normal  serum) 

To  both  of  these  after  five  hours  was  added 

Hemolytic  amboceptor 
(Heated  hemolytic  serum) 

+ 

Red  blood  cells 
Results : 

(a)  showed  no  hemolysis. 

(b)  showed  hemolysis  -f-. 

The  conclusion  to  be  drawn  from  this  was  that  in  (a)  the  presence 
of  immune  body  had  led  to  absorption  of  all  the  complement.  In 
(b),  there  being  no  bacteriolytic  immune  body  to  sensitize  the 
bacteria  and  enable  them  to  absorb  complement,  the  latter  substance 
was  left  free  to  activate  the  subsequently  added  hemolytic  ambo- 
ceptors.  The  Bordet-Gengou  phenomenon  has  been  extensively  used 
by  Wassermann  and  Bruck,54  Neisser  and  Sachs,55  and  others  to 


™Bordet  et  Gengou,  Ann.  de  Tinst.  Pasteur,  1901. 

64  Wassermann  und  Bruck,  Med.  Klin.,  1905. 

55  Neisser  und  Sachs,  Berl.  klin.  Woch.,  xliv,  1905,  and  i,  1906. 


SENSITIZING  ANTIBODIES  299 

demonstrate  the  presence  of  immune  bodies  in  various  sera.  (See 
p.  315.) 

It  should  be  noted  that  this  method,  if  valid,  must  presuppose 
the  identity  of  the  hemolytic  and  bactericidal  complement  in  the 
activating  serum. 

Complement  fixation  will  be  more  -  extensively  discussed  in  the 
section  dealing  with  the  Wassermann  reaction. 

WL.  .Complement 

I  Together  a*      4    I  I HaemolyKe 
StiphiHtic            present                                                         Aiwl>oceptor 

_  _  _  Jmmune  or     ?      or      1  37.SO  C. 

antibody  not     ^  «  « 

f  for  one  Hour 

<s.tf|— Red  blood  cell 


If  (2)   present,  no   haemolysis. 
If.  (2)   not  present,   haemolysis. 

FIG.  38. — SCHEMATIC  REPRESENTATION  OF  COMPLEMENT  FIXATION  IN  THE  BORDET- 
GENGOU  REACTION,  AS  CONCEIVED  BY  EHRLICH.  (This  scheme  is  given 
because  it  aids  in  understanding  the  process,  but  must  not  be  taken  to  repre- 
sent the  true  manner  of  union  in  which  the  complements  react.) 

The  Specificity  of  Hemolysins. — In  the  sections  preceding  we 
have  seen  that  the  blood  cells  of  one  animal,  injected  into  an  animal 
of  another  species,  give  rise  to  a  hemolytic  substance  in  the  blood 
serum  of  the  second  animal,  which  is  strictly  specific  for  the  variety 
of  cells  injected.  Such  hemolysins,  when  produced  in  one  animal 
against  blood  cells  of  another  species,  are  spoken  of  as  heterolysins. 
In  studying  the  nature  of  hemolysis,  Ehrlich  and  Morgenroth56  now 
discovered  that  hemolysins  could  also  be  produced  if  an  animal  were 
injected  with  red  blood  cells  of  a  member  of  its  own  species.  Such 
hemolytic  substances  they  called  isolysins.  In  their  experiments  they 
injected  goats  with  the  washed  red  blood  corpuscles  of  other  goats 
and  found  that  the  serum  of  the  recipient  developed  the  power  of 
causing  hemolysis  of  the  red  blood  cells  of  the  particular  goat  whose 
blood  had  been  used  for  injection.  It  did  not,  however,  possess  the 

59  Ehrlich  und  Morgenroth,  Berliner  klin.  Woch.,  xxi,  1900. 


300  INFECTION   AND  IMMUNITY 

power  of  producing  hemolysis  in  the  blood  of  all  goats,  nor  did  it 
produce  hemolysis  with  the  red  corpuscles  of  its  own  blood.  It  is 
thus  shown  that  the  specificity  of  the  hemolysins  extends  even  within 
the  limits  of  species,  and  is,  to  a  certain  extent,  an  individual  property. 

The  production  of  autolysins,  that  is,  of  substances  in  the  blood 
serum  which  will  produce  hemolysis  of  the  individual's  own  corpuscles, 
has,  so  far,  been  unsuccessful. 

Ehrlich  and  Morgenroth,  in  the  course  of  these  experiments, 
furthermore  succeeded  in  showing  that  the  injection  of  isolysins 
into  animals  produced  antiisolysins,  and  that  these  again  were 
strictly  specific. 

The  almost  universal  failure  of  autolysin  production  has  found 
no  satisfactory  explanation.  It  is  supposed  by  Ehrlich  and  Morgen- 
roth that  the  failure  of  autolysin  production  may  be  due  to  a  lack 
of  suitable  receptors  in  the  animal  for  its  own  cells. 

The  clinical  significance  of  the  presence  of  isolysins  and  possibly 
of  autolysins  in  human  beings  is  too  evident  to  require  much  dis- 
cussion. A  practical  and  extremely  interesting  result  which  these 
investigations  have  yielded  is  that  of  Donath  and  Landsteiner,57 
who  discovered  an  autolysin  in  the  blood  serum  of  patients  suffering 
from  paroxysmal  hemoglobinuria.  In  these  cases  the  sensitizing 
substance  or  amboceptor  appeared  to  be  absorbed  by  the  red  blood 
cells  only  at  low  temperatures — probably  in  the  capillaries  during 
exposure  to  the  cold,  and  hemolysis  subsequently  resulted  in  the 
blood  stream  by  the  action  of  complement.  These  observations  have 
been  confirmed  by  other  writers,  but  the  phenomenon  is  surely 
not  present  in  all  cases  of  paroxysmal  hemoglobinuria. 

Isoagglutinins  and  blood  typing  in  human  beings  are  discussed 
in  a  subsequent  section. 


57  Donath  und  Landsteiner,  Munch,  med.  Woeh.,  xxxvi,  1904. 


CHAPTER   XVI 

THE  TECHNIQUE  OF  SERUM  REACTIONS 

Obtaining  Serum  from  Animals  and  Man. — To  obtain  blood  serum 
from  man,  the  blood  may  be  taken  from  the  finger  or  the  ear,  either 
into  a  sterile  centrifuge  tube  or  into  a  Wright  capsule.  When  taken 
into  a  centrifuge  tube,  the  blood  is  allowed  to  clot  and  the  serum 
separated  by  centrifugation.  Larger  quantities  of  blood  may  be 
taken  with  a  syringe  from  the  median  basilic  vein  and  either  slanted 
in  sterile  test  tubes  in  the  ice  chest  or  put  into  centrifuge  tubes 
and  centrifugalized.  In  bleeding  small  laboratory  animals,  a  number 
of  methods  may  be  employed,  depending  upon  the  quantity  of  serum 
required. 

The  animals  most  frequently  used  for  laboratory  purposes  are 
rabbits.  To  obtain  small  quantities  of  serum  from  rabbits,  the 
animals  may  be  bled  from  the  marginal  vein  of  the  ear.  The  animal 
is  strapped  upon  a  tray  and  underneath  it  is  placed  "a  rubber  bag 
filled  with  warm  water.  This  is  advised  by  Wadsworth  to  facilitate 
the  flow  of  blood.  The  tray  is  then  placed  upon  an  easel  so  that 
the  animal's  head  hangs  downward.  The  skin  over  the  ear  vein 
is  shaved  and  sterilized,  and  a  Hagedorn  needle  plunged  into  the 
vein.  The  blood  is  caught  in  test  tubes  or  centrifuge  tubes. 

When  larger  quantities  of  blood  are  desired  it  may  be  taken  from 
the  carotid  artery.  In  rabbits,  the  carotid  may  be  found  lying  just 
lateral  to  the  trachea  and  deeply  placed,  and  must  be  carefully 
separated  from  the  pneumogastric  nerve  by  blunt  dissection.  The 
distal  end  of  the  artery  is  then  tied  off  and  the  proximal  end  tem- 
porarily closed  with  a  small  clamp.  The  artery  is  then  raised  out 
of  the  wound  on  a  knife  or  forceps  handle  and,  with  sharp-pointed 
scissors,  a  small  incision  is  made  into  but  not  through  the  vessel. 
A  small  glass  cannula  is  now  introduced  and  tied  into  place  by  a 
thread.  To  this  cannula  a  small  rubber  tube  fitted  with  a  pinch-cock 
should  have  been  attached,  the  whole  being  sterilized.  Recently 
we  have  dispensed  with  the  cannula,  simply  holding  the  vessel  up 
with  a  pointed  forceps.  A  larger  yield  of  serum  will  be  obtained 

301 


302  INFECTION  AND  IMMUNITY 

if,  after  coagulation,  the  clot  is  separated  from  the  glass  with  a 
sterile  platinum  wire. 

In  obtaining  blood  from  larger  animals,  horses,  sheep,  etc.,  a 
cannula  may  be  introduced  into  the  jugular  or  internal  saphenous 
veins.  The  skin  is  shaved  and  sterilized  and  a  rubber  tourniquet 
placed  about  the  neck  or  thigh,  as  the  case  may  be,  in  order  to 
cause  the  vein  to  stand  out.  A  small  incision  may  be  made  through 
the  skin  over  the  vein,  but  is  not  necessary.  A  cannula,  with  rubber 
tubing  attached,  is  then  plunged  into  the  vein  and  the  blood  caught 
in  sterile  high  cylindrical  jars,  allowed  to  clot,  and  placed  in  the 
refrigerator.  The  serum  is  taken  off .  after  twenty-four  to  forty- 
eight  hours  with  sterile  pipettes. 

Agglutination  Tests. — For  the  determination  of  the  agglutinating 
power  of  serum  it  is  necessary  to  make  suitable  dilutions  of  the 
serum,  and  to  prepare  an  even  emulsion  of  the  microorganisms  to 
be  tested.  The  test  may  be  made  microscopically  or  macroscopically. 
The  microscopic  test  is  the  one  in  general  use  in  the  diagnosis  of 
typhoid  fever,  and  is  occasionally  applied  to  some  other  diseases. 
In  its  application  to  typhoid  fever  it  is  usually  spoken  of  as  the 
Gruber-Widal  reaction. 

Twelve-  to  eighteen-hour  broth  cultures  of  the  typhoid  bacillus,  grown 
at  incubator  temperature,  may  be  used.  It  is  preferable,  however,  to  use 
an  emulsion  of  a  twelve  to  twenty-four  hour  old  ag'ar  culture  in  physiological 
salt  solution  (0.85  per  cent).  The  salt-solution  emulsion  is  made  by  adding 
about  10  c.c.  of  normal  salt  solution  to  the  fresh  agar  slant  culture,  carefully 
detaching  the  culture  from  the  surface  of  the  agar  with  a  flexible  platinum 
wire,  and  pipetting  off  the  emulsion  thus  made.  With  some  microorganisms 
it  is  sufficient  simply  to  allow  the  larger  clumps  to  settle  and  to  pipette 
off  the  supernatant  turbid  emulsion.  With  other  microorganisms,  the  tendency 
to  form  clumps  makes  it  necessary  to  resort  to  further  methods  of  securing 
an  even  distribution  of  the  bacteria.  This  may  be  done  either  by  sucking 
the  emulsion  in  and  out  through  a  narrow  pipette  held  perpendicularly  against 
the  bottom  of  a  watch  glass,  as  in  Wright's  technique  for  the  opsonic  test 
(see  section  on  opsonins,  p.  339),  or  by  carefully  rubbing  the  clumps  against 
the  watch  glass  with  a  stiff  platinum  wire.  In  the  case  of  the  tubercle 
bacillus  not  even  this  suffices,  but  it  becomes  necessary  to  grind  the  moist 
bacillary  masses  in  a  mortar  before  emulsifying.  With  the  tubercle  bacillus, 
too,  it  is  preferable  to  use  salt  solution  at  1.5  per  cent  concentration. 

The  serum  dilutions  are  obtained  by  first  making  a  one  to  ten 
dilution  of  serum  with  normal  salt  solution.  The  serum  used  for 


THE  TECHNIQUE  OF  SERUM   REACTIONS  303 

this  purpose  is  freed  from  red  blood  corpuscles  by  centrifugation. 
From  the  1  to  10  diluton  any  number  of  higher  dilutions  may  be  made, 
by  mixing  given  parts  of  the  1  to  10  dilution  with  normal  salt 
solution ;  thus  one  part  of  a  1  to  10  dilution  plus  an  equal  quantity 
of  salt  solution  gives  a  dilution  of  1  to  20.  One  part  of  one  to  ten 
dilution  plus  two  parts  of  normal  salt  solution  gives  one  to  thirty, 
etc.  It  must  not  be  forgotten  that,  when  equal  parts  of  the  serum 
and  bacillary  emulsion  have  been  mixed,  each  one  of  these  dilutions 
is  doubled. 

In  making  the  microscopic  agglutination  test,  equal  quantities  of 
serum  dilution  and  bacterial  emulsion  are  mixed  upon  a  cover-slip. 
The  mixture  may  be  made  either  by  measuring  out  a  drop  of  each 
substance  with  a  standard  platinum  loop,  depositing  them  close 
together  on  the  cover-slip,  and  mixing;  or  equal  quantities  may  be 
sucked  up,  each  to  a  given  mark,  in  a  capillary  pipette,  mixed  by 
suction  in  and  out,  and  deposited  upon  the  cover-slip.  The  cover- 
slip  is  inverted  over  a  hollow  glass  slide,  the  rim  of  which  has  been 
greased  with  vaseline.  The  drop  is  then  observed  through  a  (Leitz) 
No.  7  lens,  ocular  No.  3. 

Macroscopic  agglutination,  preferable  for  exact  laboratory  re- 
search, is  made  in  narrow  test  tubes  measuring  about  0.5  cm.  in 
diameter  and  about  5  cm.  in  length. 

Equal  quantities,  usually  1  c.c.  each,  of  serum  dilution  and 
emulsion  are  mixed.  A  series  of  tubes  is  prepared,  in  each  subse- 
quent one  of  which  the  dilution  is  higher.  These  mixtures  may  be 
placed  in  the  incubator  for  a  few  hours  and  then  kept  at  room 
temperature.  After  removal  from  the  incubator  agglutination  is  in 
some  instances  hastened  by  transference  to  the  ice  chest.  When 
agglutination  takes  place  in  these  tubes,  clumps  of  bacteria  may  be 
seen  to  form,  which  settle  to  the  bottom  of  the  tube,  very  much 
like  snow-flakes.  The  surface  of  the  sediment  is  heaped  up  and 
irregular.  The  supernatant  fluid  becomes  entirely  clear.  When  the 
reaction  does  not  occur  the  sediment  is  an  even,  granular  one  with, 
a  flat  surface,  and  the  emulsion  remains  turbid. 

Instead  of  using  test  tubes  as  described  above,  Wright  has  sug- 
gested the  use  of  throttle  pipettes  of  comparatively  large  diameter 
into  each  of  which  at  least  throe  or  four  different  dilutions  can  be 
sucked  with  a  nipple,  a  small  air  bubble  being  left  between,  the 
mixtures.  By  sealing  the  distal  end  of  these  pipettes  in  a  flame 
the  various  dilutions  are  kept  at  a  distance  from  each  other,  and 


304:  INFECTION  AND   IMMUNITY 

the  pipettes  may  be  set  on  end  in  a  tumbler  and  observed  just 
are  the  test  tubes  (Fig.  42,  p.  340).  The  special  methods  of  carrying 
out  agglutination  tests  with  pneumococci  have  been  described  01 
p.  460. 

Precipitin  Tests. — In  an  earlier  section  on  precipitins  we  hav< 
seen  that  precipitates  are  formed  when  clear  nitrates  of  bacterij 
extracts  or  of  both  cultures  are  mixed  with  their  specific  immune 
sera.  Such  precipitiii  reactions  are  not  limited  to  the  realm  oi 
bacteria,  but  have  a  broad  biological  significance,  in  that  specific 
precipitating  sera  may  be  produced  with  proteins  of  varied  source. 

For  carrying  out  a  precipitin  test,  the  following  reagents 
required : 

1.  A  specific  precipitating  antiserum   (antibacterial  or  antipi 
tein)  ; 

2.  A  bacterial  filtrate  or  protein  solution. 

PRODUCTION  OF  PRECIPITATING  ANTiSERA.1 — Antibacterial  pre- 
cipitins may  be  produced  in  animals  by  a  variety  of  methods. 
Animals,  preferably  rabbits,  are  injected  with  cultures  of  the  bac- 
teria in  gradually  increasing  quantities.  Five  or  six  injections  are 
given  at  intervals  of  from  five  to  six  days,  the  dosage  and  mode 
of  administration  being  adapted  in  each  case  to  the  pathogeiiicity 
of  the  microorganisms  in  question.  Myers2  claims  that  specific  pre- 
cipitin for  pepton  in  the  culture  media  may  be  formed  which  may 
lead  to  error.  This  could  not  be  confirmed  by  Norris.3  The  im- 
munized animals  should  be  bled  about  7  to  12  days  after  the  last 
injection. 

Precipitating  antisera  against  protein  solutions  are  prepared  by 
similar  methods.  Two  or  three  injections,  however,  usually  suffice. 
The  sera  or  protein  solutions  used  should  be  sterile.  This  may  be 
accomplished  by  filtration  through  small  porcelain  filters.  Injec- 
tions into  animals  may  be  made  subcutaneously,  intraperitoneally, 
or  intravenously.  The  subcutaneous  route  has  no  advantages  unless 
the  substances  to  be  used  are  contaminated.  It  is  far  easier  to 
produce  precipitating  sera  against  proteins  like  horse  serum,  egg 
albumin,  etc.,  than  it  is  to  produce  them  against  bacterial  substances. 
This  is  due  probably  to  the  fact  that  bacterial  bodies  contain  rela- 


1  E.  Kraus,  Wion.  klin.  Woch.,  1897;  Norris,  Jour.  Inf.  Dis.,  1  and  3,  1904. 
-Myers,  Lancet,  ii,  1900. 
3  Norris,  loc.  cit. 


THE  TECHNIQUE  OF  SERUM   REACTIONS  305 

lively  little  soagulable  protein,  and  the  quantity  injected,  even  with 
the  largest  tolerable  doses  of  bacteria,  may  contain  but  very  small 
amounts  of  true  protein  forming  antigen. 

The  animals  are  weighed  from  time  to  time,  and  if  considerable 
loss  of  weight  ensues,  the  intervals  should  be  increased.  Doses  from 
2  to  5  c.c.  should  be  given.  In  giving  the  later  injections  the  danger 
of  anaphylaxis  must  be  remembered.  A  single  injection  of  a  large 
quantity  has  occasionally  yielded  a  precipitating  scrum  of  consider- 
able strength,4  but  this  method  is  not  usually  successful.  Injections 
are  made  at  intervals  of  from  five  to  seven  days.  Seven  to  twelve 
days  after  the  last  injection  the  animals  may  be  bled  from  the  ear, 
and  a  preliminary  test  made  to  ascertain  the  precipitating  value  of 
the  serum.  If  this  is  insufficient,  more  injections  may  be  made. 
Bleeding  should  be  done  7  to  12  days  after  the  last  injection.  Such 
sera  may  be  preserved  in  the  dark  and  at  a  low  temperature.  If 
a  preservative  is  added,  Nuttall  prefers  chloroform  to  the  phenols, 
because  of  occasional  turbidity  produced  by  these.  Fornet  and 
Miiller5  and  others  have  recommended  rapid  methods  of  precipitin 
formation  by  injecting  relatively  large  amounts  of  the  antigen  daily, 
for  three  or  four  successive  days,  bleeding  on  the  fifth  or  sixth  day 
thereafter.  This  method  has  been  followed  by  a  number  of  workers 
subsequently,  and  is  often  successful,  sera  of  considerable  titers 
being  obtained.  In  our  experience,  however,  this  method  has  not 
shown  itself  to  be  entirely  advantageous,  since  it  is  rare  that  a  very 
potent  serum  is  so  produced,  and  also  animals  bleed  within  a  week 
after  repeated  large  doses,  may  show  in  their  serum  not  only  anti- 
bodies but  also  residues  of  antigen,  the  two  substances  not  united 
within  the  animal  body,  but  gradually  uniting  and  forming  precipi- 
tates after  the  serum  has  been  obtained  and  stored. 

This  phenomenon  of  the  simultaneous  presence  of  antigen  and 
antibody  in  the  circulating  blood  has  been  variously  explained,  the 
view  formerly  held  being  that  of  von  Dungern,  who  believed  that 
every  injected  antigen  contained  partial  antigens,  a,  b,  c,  etc.,  each 
of  which  produced  its  partial  antibody,  A,  B,  C,  which,  by  being 
present  in  unequal  proportions,  gave  rise  to  the  simultaneous 
presence  of  perhaps  a  and  B,  etc.  Our  own  opinion  is6  that  the 


*Michaelis,  Deut.  med.  Woch.,  1902. 

5  Fornet  and  Muller,  Zeit.  f.  Hyg.,  66,  1910. 

6  Zinsser  and  Young,  Jour.  Exper.  Med.,  17,  1913. 


306  INFECTION   AND   IMMUNITY 

failure  of  union  in  the  circulating  blood  of  the  living  animal  is  due 
to  actions  analogous  to  those  of  protective  colloids,  and  represent 
a  protective  mechanism  which  represents  the  sudden  union  of  anti- 
gen and  antibody  in  the  circulation. 

Precipitating  antisera  for  tests  should  be  clear.  If  turbid,  the 
sera  should  be  filtered  through  small  Berkefeld  or  porcelain  candles. 

PREPARATION  OF  BACTERIAL  FILTRATES  AND  PROTEIN  SOLUTIONS  FOB 
PRECIPITIN  TESTS. — Bacteria  may  be  grown  in  nutrient  broth  having 
an  initial  reaction  of  neutrality  or  five-tenths  per  cent  acidity  to 
phenolphthalein.  The  cultures  are  incubated  for  times  varying 
from  a  week  to  several  months,  and  are  then  filtered  through  por- 
celain or  Berkefeld  candles  until  perfectly  clear.  Bacterial  extracts 
may  also  be  made  by  emulsifying  agar  cultures  in  salt  solution, 
placing  at  37.5  C.  in  the  incubator  for  a  week  or  longer,  and  filter- 
ing. More  rapid  extraction  of  bacteria  may  be  accomplished  by 
repeated,  rapid  freezing  and  thawing  of  salt-solution  emulsions,  by 
shaking  in  the  shaking  machine  or  by  centrifugalizing,  rubbing  up 
the  sediment  with  dry  salt,  and  the  addition  of  distilled  water  to 
isotonicity. 

Protein  solutions  to  be  tested  should  be  made  in  salt  solution. 
When  dealing  with  blood  stains,  as  in  doing  the  test  for  forensic 
purposes,  the  stains  should  be  dissolved  in  salt  solution,  an  ap- 
proximate dilution  of  one  in  five  hundred  being  aimed  at.  This 
solution  if  turbid  should  be  filtered  through  a  small  porcelain  filter. 
It  should  be  clear  and  colorless,  show  a  faint  cloud  on  boiling  with 
dilute  acetic  acid,  and  show  distinct  froth  when  shaken. 

When  the  reaction  is  to  be  done  for  determining  the  nature  of 
meat  (detection  of  horse-meat  substitution  for  beef,  etc.),  about  20 
to  40  grams  of  the  suspected  meat  are  macerated  in  a  flask,  and 
covered  with  100  c.c.  of  salt  solution.  This  mixture  is  allowed  to 
infuse  at  room  temperature  for  three  to  four  hours,  and  is  then 
placed  in  the  refrigerator  for  twelve  hours  or  more.  At  the  end 
of  this  time  2  c.c.  are  shaken  into  a  test  tube.  If  profuse  frothing7" 
appears,  the  extract  is  ready  for  use.  It  is  then  filtered  clear,  either 
through  paper,  or  through  a  Buchner  or  Nutsche  filter.  Berkefeld 
filters  may  also  be  used.  The  solution  is  then  diluted  until  the 
addition  of  concentrated  IIN03  produces  only  a  slight  even  turbidity. 


7  P.  Th.  Milller,  ' '  Technik.  d.  serodiagnos.  Methoden. ' ' 


THE   TECHNIQUE  OF  SERUM   REACTIONS  307 

Before  use  the  reaction  of  the  meat  extract  should  be  tested,  and 
if  necessary  adjusted  to  neutrality  or  slight  acidity  or  alkalinity. 

In  the  actual  test  with  bacterial  filtrate,  the  procedure  is  as 
follows:  In  a  series  of  narrow  test  tubes,  the  following  mixtures 
are  made: 

Tube  1.  Antibacterial  serum  .5  c.c.  -f  bacterial  nitrate  1.  c.c. 

"  2.  Normal  serum  .5  c.c.  +  bacterial  filtrate  1.  c.c. 

"  3.  Antibacterial  serum  .5  c.c.  +  salt  solution          1.  c.c. 

"  4.  Salt  solution  .5  c.c.  -f-  bacterial  filtrate  1.  c.c. 

Place  the  tubes  in  the  incubator  at  37.5°  C.  Tube  1  only  should 
show  a  haziness  which  develops  into  distinct  cloudiness  or  a  floc- 
culent  precipitate  within  one  hour.  Tubes  2,  3,  and  4  should  remain 
clear. 

In  testing  an  unknown  protein  with  serum  of  an  animal  im- 
munized with  the  protein  sought  for,  the  technique  of  the  test  is 
as  follows: 

1.  0.1  c.c.  immune  serum  +  2  c.c.  unknown  protein  solution. 

2.  0.1  c.c.  immune  serum  +  2  c.c.  known  protein  solution  of  variety  sus- 

pected (similarly  diluted). 

3.  0.1  c.c.  immune  serum  +  2  c.c,  protein  solution  of  different  nature 

(similarly  diluted). 

4.  0.1  c.c.  immune  serum  +  2  c.c.  salt  solution. 

5.  2  c.c.  unknown  protein  solution. 

If  the  test  is  positive  a  precipitate  appears  in  tubes  1  and  2,  but 
not  in  any  of  the  others.  The  precipitate  should  appear  within 
15  to  20  minutes. 

Bactericidal  and  Bacteriolytic  Tests.— The  bactericidal  and  bac- 
teriolytic powers  of  serum  may  be  tested  either  in  the  animal  body 
or  in  the  test  tube.  The  in  vivo  test  is  known  as  Pfeiffer's 
phenomenon.  This  depends  upon  the  fact  that  bacteria,  when  in- 
jected into  the  peritoneal  cavity  of  a  guinea-pig,  together  with  a 
homologous  immune  serum,  undergo  dissolution. 

As  practiced,  the  test  finds  a  double  application.  It  may  be  done 
to  determine  the  bacteriolytic  power  of  a  given  serum  against  a 
known  microorganism,  or  for  the  identification  of  a  particular  micro- 
organism by  means  of  its  susceptibility  to  lysis  in  a  known  immune 
serum. 


308  INFECTION   AND   IMMUNITY 

1.  Determination  of  tJie  bacteriolytic  power  of  serum  against  a 
known  microorganism  in  vivo:* 

A  number  of  dilutions  of  the  serum  are  made  with  sterile  neutral 
bouillon  or  salt  solution,  ranging  from  1  in  20  to  1  in  500,  or  higher. 
It  is  convenient  to  make  a  first  solution  of  1  in  20.  One  c.c.  of 
this  mixed  with  4  c.c.  of  broth  will  give  1  in  100.  One  c.c.  of  the 
1  in  100  dilution  with  1  c.c.  of  broth,  2  c.c.  of  broth  and  4  c.c.  of 
broth  will  give  1  in  200,  1  in  300,  and  1  in  500  respectively.  Into 
one  cubic  centimeter  of  each  of  these  dilutions  there  is  placed  one 
platinum  loopful  of  a  twenty-four-hour  agar  culture  of  the  micro- 
organism against  which  the  serum  is  to  be  tested.  Into  another  test 
tube  is  placed  4  c.c.  of  broth,  without  serum,  and  with  one  loopful 
of  the  microorganisms.  The  mixtures  are  thoroughly  emulsified  in 
each  case  by  rubbing  the  bacteria  against  the  sides  of  the  tube  with 
the  platinum  loop. 

Intraperitoneal  injections  into  guinea-pigs  are  then  made  of  1  c.c. 
of  each  of  the  serum-dilution-bacterial-emulsions.  A  control  guinea- 
pig  (better  two  or  three)  receives  1  c.c.  of  the  broth  emulsion— 
one-fourth  as  many  bacteria,  therefore,  as  the  animals  receiving  the 
serum  dilutions. 

Before  making  the  injections,  areas  on  the  lateral  abdominal 
walls  of  the  guinea-pigs  are  shaved,  and  small  incisions  made  through 
the  skin,  down  to  the  muscular  layers.  The  needle  of  the  syringe 
is  then  introduced  perpendicular  to  the  skin  until  it  has  penetrated 
the  peritoneum,  and  then  carefully  slanted  to  avoid  puncturing  the 
gut,  The  animals  need  not  be  strapped  down  during  this  procedure 
and  afterward  may  be  allowed  to  run  about. 

After  one-half  hour,  and  again  after  one  hour  has  elapsed,  a  drop 
of  peritoneal  exudate  is  removed  from  each  guinea-pig  and  examined 
in  the  hanging  drop  for  granulation  and  swelling  of  the  bacteria. 
The  method  of  obtaining  the  peritoneal  exudate  is  as  follows :  Small 


FIG.  39. — CAPILLARY  PIPETTE  FOR  REMOVAL  OF  EXUDATE  IN  DOING  THE  PFEIFFER 

TEST. 

glass  tubing  is  drawn  out  into  capillary  pipettes,  the  ends  of  the 
capillaries  being  again  drawn  to  fine  points  in  a  small  yellow  flame. 

8  P.  Th.  Muller,  "Technik  d.  serodiagnos.  Methoden,"  Jena,  1909. 


THE  TECHNIQUE  OF  SERUM  REACTIONS  309 

A  number  of  such  pipettes  should  be  prepared  before  the  test  is 
begun.  The  guinea-pig  is  then  held  down  upon  a  table,  either  by 
an  assistant  or  by  the  left  hand  of  the  operator,  and  the  point  of 
the  pipette  pushed  through  the  cut  in  the  abdominal  wall  into  the 
peritoneum  by  a  sharp,  quick  thrusting  motion.  A  column  of  peri- 
toneal fluid  will  run  into  the  glass  tubing  by  capillary  attraction; 
this  can  then  be  blown  out  upon  a  cover-slip  for  hanging-drop 
examination  or  may  be  blown  upon  a  slide,  smeared,  and  examined 
after  staining.  The  reaction  is  regarded  as  positive  if  within  thirty 
minutes  to  an  hour  the  peritoneal  exudates  of  the  animals  receiving 
immune  sera  contain  only  swollen  or  disintegrated  microorganisms, 
while  in  that  of  the  control  animals  only  well-preserved  and  unde- 
generated  bacteria  are  found.  In  dealing  with  typhoid  bacilli  and 
cholera  spirilla,  in  connection  with  which  the  test  is  most  often 
used,  active  motility  in  the  controls  is  of  much  help.  Should  there 
be  extensive  degeneration  of  the  bacteria  in  the  exudate  of  the  con- 
trol animals  the  test  is  of  no  value. 

2.  Identification  of  a  microorganism  by  observing  its  susceptibility 
to  lysis  in  a  known  immune  serum  in  vivo: 

The  technique  for  this  test  is  practically  the  same  as  that  of 
the  preceding  except  that  in  this  case  we  require  a  potent  known 
immune  serum  and  normal  serum  for  control.  It  is  necessary, 
furthermore,  that  by  previous  tests  we  should  know  the  degree  of 
dilution  in  which  the  immune  serum  will  cause  complete  bacteriolysis 
of  the  microorganism  used  in  its  production.  Thus,  if  we  are  em- 
ploying a  typhoid  immune  serum  and  are  about  to  test  by  this 
method  an  unknown  Gram-negative  bacillus,  we  must  know  the 
titer  of  the  serum  for  the  typhoid  bacillus  itself. 

Mixtures  are  then  made  of  dilutions  of  this  serum  and  definite 
quantities  of  the  microorganism  to  be  tested.  It  is  best,  always, 
to  employ  from  ten  to  one  hundred  times  the  amount  of  immune 
serum  which  suffices  to  produce  lysis  with  its  homologous  micro- 
organism. Thus,  if  the  serum  has  been  found  to  be  active  in  dilu- 
tions of  1:1,000,  it  is  employed  in  the  test  in  dilutions  at  1:1,000, 
1 :100,  and  1 :10.  These  dilutions  are  then  injected  into  guinea-pigs 
in  quantities  of  1  e.c.  together  with  the  bacteria  to  be  tested,  and 
control  guinea-pigs  are  injected  with  undiluted  normal  serum  mixed 
with  the  bacteria  and  with  salt  solution  and  the  bacteria.  The 
exudates  are  then  observed  in  the  same  way  as  in  the  preceding 
experiment. 


310 


INFECTION   AND   IMMUNITY 


Bactericidal  Reactions  in  tlie  Test  Tube. — Bactericidal  reactions 
in  the  test  tubes  may  be  made  by  mixing  in  small  sterile  test  tubes, 
definite  quantities  of  the  bacteria  with  inactivated  serum  and  com- 
plement, the  latter  in  the  form  of  unheated  normal  serum.  The 
mixtures,  diluted  with  equal  volumes  of  neutral  broth  or  salt  solu- 
tion, are  set  away  for  a  definite  time  three  to  four  hours  in  an 
incubator  at  37.5°  C.,  and  equal  quantities  from  all  the  tubes  are 
then  inoculated  into  melted  agar  at  40°  C.,  and  plates  are  poured. 
Control  plates  must  be  made  in  each  case  with  mixtures  of  similar 
quantities  of  bacteria  in  salt  solution,  and  similar  quantities  of  bac- 
teria in  normal  serum.  By  colony  counting  after  the  plates  have 
developed,  it  is  then  possible  to  estimate  the  degree  of  bacterial 
destruction  in  any  of  the  given  dilutions. 

In  actually  carrying  out  the  test,  dilutions  of  the  inactivated 
serum  are  first  made,  ranging  from  1 :10  to  1 :1,000  and  over.  An 
emulsion  of  bacteria  from  a  twenty-four-hour  agar  slant  is  then 
made  in  salt  solution,  or  a  twenty-four-hour  broth  culture  properly 
diluted  may  be  used.  Complement  is  obtained  by  taking  fresh 
normal  rabbit  serum  and  diluting  it  with  salt  solution  1 :10  or  1 :15. 
Into  a  series  of  test  tubes,  then,  1  c.c.  of  each  of  the  serum  dilutions 
is  placed,  and  to  each  tube  is  added  0.5  c.c.  of  the  diluted  fresh 
normal  rabbit  serum  (complement).  To  these  mixtures  the  bacteria 
are  then  added.  In  adding  the  bacterial  emulsion  to  these  tubes, 
the  writers  have  found  it  more  accurate  to  discard  the  use  of  the 
platinum  loop  and  to  measure  the  bacterial  emulsion  in  a  marked 
capillary  pipette  such  as  that  used  in  the  opsonin  test.  (See  page 
340,  Fig.  42.)  The  controls  are  set  up  in  a  similar  way,  all  of  them 
containing  a  similar  quantity  of  bacterial  emulsions,  one  control 
containing  1.5  c.c.  of  salt  solution,  another  control  containing  1  c.c. 
of  salt  solution  -f-  0.5  c.c.  of  the  diluted  complement,  and  the  third 
control  containing  inactivated  normal  serum  1  c.c.  -f-  0.5  c.c.  of 
diluted  complement.  Definite  quantities  of  these  mixtures,  taken 
with  a  standard  loop,  or  preferably  with  a  capillary  pipette,  are 
plated  in  agar  immediately  after  mixing. 

After  incubation  for  two  or  three  hours  similar  quantities  are 
again  measured  into  tubes  of  melted  agar  with  the  capillary  pipette. 
With  a  little  practice,  great  accuracy  in  these  measurements  can. 
be  acquired.  The  inoculated  agar  tubes  arc  very  thoroughly  mixed, 
and  plates  are  poured.  At  the  end  of  twenty-four  hours'  incubation, 


THE   TECHNIQUE   OF  SERUM   REACTIONS 


311 


an  enumeration  of  the  colonies  in  the  various  plates  is  made  and 
the  results  are  compared. 

BACTERICIDAL  TEST  IN  VITRO 

(To  DETERMINE  THE  BACTERICIDAL  POWER  OF  A  TYPHOID  IMMUNE  SERUM  AGAINST 

TYPHOID  BACILLI). 


Plates 

Poured 

After  3  Hrs. 

at  37°  C. 

1  c.c.  Immune  Typh.  Ser.  1:200     +0.5  c.c.  Typh.  Emu 
1    "                                         1:400     +0.5    " 

lsion+0.5c.c.  Rab.  S 
+  0.5    " 

er.  1 
1 

:  15  \          0 
15    /    Colonies. 

1 
1 
1 

"     1:800     +0.5 
"     1:1600   +0.5 
"     1:3200   +0.5 

+  0.5    ' 
+  0.5    ' 
+0.5    ' 

1 

}£  \  100-1,000 
{£  J    Colonies. 

1 

1 

1 

•          ' 

"      1:6400   +0.5 

+  0.5    ' 

*  ' 

1 

15  1    More  than 

1 
1 

• 

"     1:25600  +  0.5 

+  0.5 
+0.5    ' 

" 

1 
1 

15   f       10,000 
15  J    Colonies 

lib  I  1.0 


1.5  c.c.  NaCl  +  0.5  Typh.  Emulsion 
1.5    "        "      +0.5 


CONTROLS 


+  0.5 


+  0.5  c.c.  Rab.  Ser.  1:15 


Plated  immediately  ]   More  than 
after  3  hrs.     \      10,000 
"     3    "       J    Colonies. 


The  in  vitro  bactericidal  tests  have  been  employed,  practically, 
chiefly  in  the  diagnosis  of  typhoid  fever  by  Stern  and  Korte.9  While 
the  serum  of  normal  individuals  shows  practically  no  bactericidal 
power  for  typhoid  bacilli,  the  sera  of  typhoid  patients  may  be  ac- 
tively bactericidal  in  dilutions  as  high  as  1 :50,000. 

Protection  tests  in  mice,  etc.,  are  described  under  the  "  standard- 
ization" of  pneumococcus  and  streptococcus  serum  in  the  chapters 
dealing  with  these  organisms. 

Hemolytic  Tests.— Determination  of  the  hemolytic  action  of  blood 
serum,  bacterial  filtrates,  and  of  a  variety  of  other  substances,  such 
as  tissue  extracts  and  animal  and  plant  poisons,  is  frequently  made 
in  bacteriological  laboratories.  Familiarity  with  the  methods  of 
carrying  out  such  tests  is  especially  essential  since  hemolytic  tests 
are  also  employed  in  determining  other  serum  reactions,  such  as 
the  "complement-fixation  tests"  discussed  in  another  section. 

For  these  tests  it  is  necessary  to  prepare  washed  red  corpuscles 
of  the  species  of  animal  against  which  the  hemolysins  are  to  be 
tested,  and  to  obtain  these,  blood  may  be  taken  in  one  of  the  follow- 
ing ways: 

A.  If  small  quantities  of  blood  corpuscles  are  desired,  the  blood 
may  be  received  into  a  sterile  test  tube  into  which  a  copper  or  other 
wire  bent  into  a  loop  at  the  lower  end  has  been  introduced.  This 


9  Stern  und  Korte,  Berl.  klin.  Woch.,  1904. 


312  INFECTION  AND  IMMUNITY 

is  used  to  prevent  clotting  and  to  remove  the  fibrin.  Immediately 
after  receiving  the  blood  into  this  tube,  the  wire  is  twirled  between 
the  fingers  so  that  the  blood  is  beaten  by  the  wire  as  by  an  egg- 
beater.  At  the  end  of  five  minutes  of  continuous  agitation,  the 
fibrin  adhering  in  a  mass  to  the  wire  may  be  lifted  out.  The  cor- 
puscles are  then  washed  and  centrifugalized  in  several  changes  of 
salt  solution  to  remove  all  traces  of  serum,  and  are  finally  emulsified 
in  salt  solution. 

B.  The  blood  may  be  taken  into  a  centrifuge  tube  and  imme- 
diately centrifugalized  before  clotting  has  taken  place.    The  plasma 
is  then  poured  off  and  the  corpuscles  are  washed  with  salt  solution, 
as  before,  to  remove  the  serum. 

C.  The  blood  may  be  taken  directly  into  a  solution  containing 
five-tenths  per  cent  sodium  chlorid  and  one  per  cent  sodium  citrate. 
The  corpuscles  are  concentrated  by  centrifugalization,  the  citrate 
solution  is  decanted,  and  corpuscles  are  washed  with  salt  solution, 
as  before,  to  remove  the  serum. 

D.  When  large  quantities  of  blood  are  desired,  either  from  man 
or  from  an  animal,  the  blood  may  be  received  directly  into  a  flask 
into  which  a  dozen  or  more  glass  beads  or  short  pieces  of  glass 
tubing  have  been  placed.    The  flask  is  shaken  for  five  or  ten  minutes, 
immediately  after  the  blood  has  been  taken  and,  in  this  way,  de- 
fibrination  is  accomplished. 

Since,  for  comparative  tests,  it  is  necessary  to  establish  some 
standard  concentration  of  red  blood  cells,  it  is  customary  in  these 
tests  to  employ  a  five  per  cent  emulsion  of  corpuscles  in  salt  solution. 
To  obtain  this,  one  volume  of  sediment  of  washed  red  blood  cells 
is  mixed  with  nineteen  parts  of  0.85  per  cent  salt  solution.10  Such 
an  emulsion,  if  kept  sterile  and  in  the  refrigerator,  will  serve  for 
hemolytic  tests  for  from  one  to  three  days.  An  emulsion  should 
not  be  used  if  the  supernatant  salt  solution  shows  any  transparent 
redness,  as  this  indicates  hemolysis. 

If  the  substance  in  which  hemolysins  are  to  be  determined  is 
serum,  this  should  be  inactivated  by  exposure  to  56°  C.  in  a  water 
bath,  and  to  each  test,  complement  may  be  added  in  the  form  of 
fresh  guinea-pig  or  rabbit's  serum.  No  absolute  rule  for  the  quan- 


10  The  method  here  given  was  formerly  much  employed.  It  is  now  the  general 
practice,  however,  to  use  one  volume  of  the  actual  sediment  to  nineteen  volumes 
of  salt  solution. 


THE  TECHNIQUE  OF  SERUM  REACTIONS  313 

tity  of  complement  to  be  used  in  these  tests  can  be  given.  In  each 
case  the  particular  complement  used  should  be  titrated  to  determine 
the  minimum  quantity  which  will  produce  hemolysis  of  1  c.c.  of  the 
sensitized  cell  suspension. 

In  the  actual  test,  mixtures  are  made  of  the  corpuscle  emulsion, 
the  inactivated  immune  serum,  and  complement  in  small  test  tubes 
and  the  volumes  of  the  various  tubes  made  equal  by  the  addition 
of  definite  quantities  of  salt  solution.  The  contents  of  the  tubes 
are  thoroughly  mixed  and  the  tubes  put  in  the  incubator  or  in  a 
water  bath  at  37.5°  C.  If  complete  hemolysis  occurs,  the  fluid  in 
the  tube  will  assume  a  deep  Burgundy  red.  If  no  hemolysis  occurs, 
the  fluid  will  remain  uncolored  and  the  corpuscles  will  settle  out. 
Incomplete  hemolysis  will  be  evidenced  by  a  lighter  tinge  of  red 
in  the  tube  and  the  settling  out  of  a  varying  quantity  of  blood 
corpuscles. 

In  all  hemolytic  tests  the  time  element  is  important.  No  hemolysis 
should  be  adjudged  as  incomplete  unless  at  least  one  hour  has 
elapsed. 

ISOAGGLUTININS 

In  1901  Landsteiner  found  that  22  individuals  whose  blood  he 
studied  could  be  divided  into  three  groups  with  respect  to  iso- 
hemoagglutinins.  It  was  found,  in  other  words,  that,  analogous  to 
the  isolysins  described  by  Ehrlich  and  his  co-workers  in  the  case 
of  goats,  human  beings  could  exert  specific  hemoagglutinating  action, 
and,  in  some  cases,  hemolytic  action  upon  the  corpuscles  of  other 
individuals.  This  is,  of  course,  of  the  greatest  importance  in  con- 
nection with  transfusion  tests.  It  is  hardly  worth  while  to  go  into 
detailed  historic  considerations  in  this  place.  Subsequent  investiga- 
tion has  shown  that  there  are  four  main  isoagglutinating  groups 
among  human  beings.  The  first  classification  of  this  kind  was  made 
by  Jansky.  Subsequently,  a  similar  classification  was  made  by  Moss, 
but  unfortunately  Moss  reversed  the  tabulation  in  such  a  way  that 
Jansky 's  group  I  became  Moss's  group  IV,  and  vice  versa.  In 
America  the  Moss  classification  has  become  universal,  and,  for  this 
reason,  the  table  given  below  represents  the  Moss  classification.  It 
must  be  borne  in  mind  by  all  workers  who  control  human  transfu- 
sions on  this  test,  however,  that  such  a  reversal  of  Moss  and  Jansky 
exists,  and  whenever  report  is  rendered  it  must  be  made  entirely 


314 


INFECTION  AND  IMMUNITY 


plain  which  classification  is  being  referred  to,  and  the  serologist 
ought  to  see  to  it  that  no  error  arises  from  misunderstanding. 

The  table  indicates  also  that  the  blood  groups  can  be  explained 
by  the  existence  of  two  agglutinins,  a  and  /?,  and  two  a'gglutinogens, 
A  and  B.  How  these  may  determine  the  reaction,  the  table  makes 
clear. 

SERA 


o 


I 

II 

III 

IV 

0 

a 

* 

•* 

I 

AB 

+ 

+ 

+ 

II 

B 

III 

A 

+ 

+ 

IV 

0 

Group  I  constitutes  about  8  per  cent  of  human  beings.  This 
group  is  generally  said  to  be  capable  of  receiving  blood  from  group 
I,  II,  III,  and  IV,  but  may  be  usedv  as  a  donor  only  for  group  I. 
It  is  spoken  of  as  a  universal  recipient,  although  it  is  best  to  avoid 
this  and  use  the  same  group,  if  possible. 

Group  II  constitutes  about  40  per  cent  of  all  persons,  and  may 
be  used  as  a  donor  for  groups  I  and  II,  or  may  receive  from  group 
II  and  IV. 

Group  III,  about  10  or  more  per  cent,  may  be  used  as  donor 
for  group  I  and  III,  or  may  receive  from  groups  III  and  IV. 

Group  IV,  about  50  per  cent  of  individuals,  may  receive  from 
group  IV  only,  but  is  spoken  of  as  the  " universal  donor."  How- 
ever, recent  writers,  especially  linger,  have  warned  against  the  use 
of  the  so-called  "universal  donor"  since  severe  reactions  have  re- 
sulted following  the  use  of  group  IV  donors  for  people  of  another 
group. 

In  all  cases  it  is  always  best  to  try  to  get  a  donor  of  the  same 
group. 


THE  TECHNIQUE  OF  SERUM   REACTIONS  315 

The  tests  may  be  done  in  a  number  of  different  ways;  the  most 
easily  carried  out,  however,  is  the  so-called  slide  agglutination  which 
was  introduced  during  the  war. 

In  order  to  properly  type  an  unknown  blood,  sera  from  group 
II  and  III  should  be  available.  A  drop  of  each  of  these  sera  is 
put  upon  a  slide  and  the  corpuscles  of  the  unknown  blood  added 
either  in  the  form  of  a  fraction  of  a  drop  of  the  blood  taken  directly 
from  the  finger  or  ear  of  the  subject,  or,  better,  a  fraction  of  a  drop 
of  defibrinated  blood  or  blood  taken  into  about  twice  its  volume 
of  salt  solution  or  sodium  citrate  solution.  The  preparation  of  the 
blood  to  be  tested  is  so  simple  that  nothing  further  need  be  said. 
The  corpuscles  so  obtained  are  mixed  with  the  types  of  sera  of 
types  II  and  III.  By  referring  to  the  table  it  will  be  easily  seen 
that,  if: 

The  blood  agglutinates  in  neither  of  the  sera,  the  subject  belongs 
to  type  IV. 

If  the  cells  agglutinate  in  type  II,  and  not  in  type  III  serum, 
the  subject  belongs  to  type  III. 

If  the  corpuscles  agglutinate  in  type  III  and  not  in  type  II 
serum,  the  subject  belongs  to  type  II. 

If  the  corpuscles  agglutinate  in  both  sera,  the  subject  belongs 
to  type  I. 

The  blood  types  as  described  above  are  among  the  few  serological 
reactions  which  are  inheritable  by  Mendelian  laws.  They  are  not, 
however,  present  in  the  child  at  birth.  According  to  recent  studies 
by  Unger,  only  about  25  per  cent  of  new  born  infants  have  cells 
that  can  be  agglutinated.  And  only  about  13  per  cent  of  new  born 
children  have  isoagglutinins.  Incompatibility  between  mother  and 
child  may  occur. 

The  Determination  of  Antibodies  in  Sera  by  Complement  Fixa- 
tion.— The  principle  of  complement  fixation,  discovered  by  Bordet 
and  Gengou11  in  1901,  has  been  utilized  both  in  bacteriological  inves- 
tigations, and  in  practical  diagnosis  for  the  determination  in  serum 
of  the  presence  of  specific  antibodies.  The  reaction  depends  upon 
the  fact  that  when  an  antigen,  i.e.,  a  substance  capable  of  stimulating 
the  formation  of  antibodies,  is  mixed  with  its  inactivated  antiserum, 
in  the  presence  of  complement,  the  complement  is  fixed  by  the  com- 
bined immune  body  and  antigen  and  can  no  longer  be  found  free  in  the 

11  Bordet  and  Gengou,  Ann.  de  1'inst.  Pasteur,  xv,  1901. 


316  INFECTION   AND   IMMUNITY 

mixture.  If  such  a  mixture  is  allowed  to  stand  at  temperature  for 
an  hour  or  more,  and  to  it  is  then  added  an  emulsion  of  red  blood 
cells  together  with  inactivated  hemolytic  serum,  no  hemolysis  will 
take  place,  since  there  is  no  free  complement  to  complete  the  hemolytio 
system.  If,  on  the  other  hand,  the  original  mixture  contains  no  anti- 
body for  the  antigen  used,  the  complement  present  is  not  fixed  and 
is  available  for  the  activation  of  the  hemolytic  serum  later  added. 

The  reaction  thus  depends  upon  the  fact  that  neither  antigen  1S 
alone,  nor  amboceptor  (antibody)  alone,  can  fix  complement,  but 
that  this  fixation  is  carried  out  only  by  the  combination  of  antigen 
plus  amboceptor.  Any  specific  can  be  determined  by  this  method, 
provided  the  homologous  antigen  is  used ;  and  vice  versa,  by  the  use  of 
a  known  antibody  a  suspected  antigen  may  be  determined. 

When  testing  immune  sera  for  antibodies  given  rise  to  in  man  or 
animals  by  microorganisms  which  can  be  cultivated,  either  the  whole 
bacteria  or  extracts  of  the  bacteria  may  be  used  as  an  antigen. 

For  the  diagnosis  of  syphilis  by  this  method,  in  the  so-called 
"Wassermann  reaction, "  the  antigen  employed  was  originally  ob- 
tained by  the  extraction  of  syphilitic  organs,  in  which  free  syphilitic 
antigens,  i.e.,  uncombined  products  of  Spirochaete  pallida,  were 
assumed  to  be  present. 

It  has  been  more  recently  shown,  however,  that  the  Wassermann 
reaction  is  not  specific  in  any  sense  of  the  word  and  that  suitable 
antigens  can  be  produced  by  the  alcoholic  extraction  of  lipoids  from 
the  normal  organs  of  many  animals  and  man. 

Bacterial  extracts  for  complement-fixation  can  be  made  in  various 
ways.  The  use  of  thick  salt  solution  suspensions  of  the  cultures  them- 
selves is  not  advisable  because  of  the  anticomplementary  action  of  such 
suspensions.  Good  bacterial  antigens  can  be  produced  by  centrifugal- 
izing  them  from  salt  solution  suspensions  and  adding  to  about 
20  mgms.,  90  mgms.  of  common  salt,  rubbing  up  with"  a  glass  rod 
for  an  hour,  and  then  adding  distilled  water  to  isotonicity.  This  is 
the  method  of  Besredka.  This  method  has  been  used  with  success  by 
Miller  and  Zinsser  in  the  case  of  tubercle  bacilli  for  complement- 
fixation  in  tuberculosis. 

Wassermann  and  Bruck  1S  prepare  bacterial  antigen  by  emulsifying 


12  Bordet  and  Gay,  Ann.  de  1  'inst.  Pasteur,  xx,  1906. 

13  Wassermann  und  Bruck,  Med.  Klinik,  55,  1905,  and  Deut.  med.  Woch.,  xii, 
1906. 


THE  TECHNIQUE  OF  SERUM   REACTIONS  317 

growths  of  about  ten  agar  slant  cultures  in  10  c.c.  of  sterile,  distilled 
water.  This  is  shaken  for  twenty-four  hours  in  a  shaking  apparatus. 
At  the  end  of  this  time  0.5  per  cent  of  carbolic  acid  is  added  and  the 
fluid  cleared  by  centrifugalization. 

The  Wassermann  Test  for  the  Diagnosis  of  Syphilis.14— The  sub- 
stances for  the  test  are  the  following : 

I.  The  Antigen. — In  their  original  experiments,  Wassermann  and 
his  collaborators  made  use  of  salt-solution  extracts  of  the  organs 
(chiefly  of  the  spleen)  of  a  syphilitic  fetus.  The  tissue  was  cut  into 
small  pieces  and  to  one  part  by  weight  of  this  substance,  four  parts  of 
normal  salt  solution  and  0.5  per  cent  of  carbolic  acid  were  added.  This 
was  shaken  in  a  shaking  apparatus  for  twenty-four  hours,  and  after 
this  the  coarser  particles  removed  by  centrifugalization.  The  reddish 
supernatant  fluid  was  used  as  the  antigen  and  could  be  preserved  for 
a  long  time  in  dark  bottles  in  the  ice  chest. 

Alcoholic  extracts  of  syphilitic  organs  were  subsequently  used  by 
a  number  of  authors,  syphilitic  liver  being  extracted  for  twenty-four 
hours  with  five  times  the  volume  of  absolute  alcohol.  This  was  filtered 
through  paper  and  the  alcohol  evaporated  in  vacuo  at  a  temperature 
not  above  40°  C.  About  1  gram  of  this  material  was  then  emulsified 
in  100  c.c.  of  salt  solution  to  which  0.5  per  cent  of  carbolic  acid  has 
been  added. 

It  was  soon  found  that  the  Wassermann  antigen  was  a  purely  non- 
specific substance,  and  since  this  discovery  was  made,  there  are  few 
laboratories  in  which  syphilitic  organs  are  still  used.  It  appears 
that  lipoidal  extracts  from  almost  any  tissue  can  be  employed,  and 
that  fairly  useful  antigens  can  even  be  obtained  with  solutions  of 
commercial  lecithin  and  mixtures  of  commercial  lecithin  and  sodium 
oleate.  It  is  apparent,  therefore,  that  in  the  Wassermann  reaction  an 
even  suspension  of  lipoidal  substances  constitutes  the  antigen,  and 
that  the  complement-fixing  complex  is  made  by  these  antigens  in  com- 
bination with  some  substance  spoken  of  by  Noguchi  as  *  *  lipotrophic ' '  in 
the  syphilitic  serum,  which  has  probably  no  relation  to  true  antibody. 
Our  own  work  with  treponema  pallidum  antigen  would  tend  to  con- 
firm this,  as  well  as  the  experience  of  Noguchi,  Craig  and  Nichols, 
Kolmer,  and  others,  who  have  found  that  a  pure  treponema  pallidum 
extract  gives  reactions  in  only  a  few  late  tertiary  cases,  running  not 


14  Wassermann,  Neisser  und  Bruck,  Deut.  med.  Woch.,  xix,  1906;  Wassermann, 
Neisser,  Bruck  und  Schucht,  Zeit.  f.  Hyg.,  lv.  1906. 


318 


INFECTION  AND   IMMUNITY 


at  all  parallel  to  the  fixations  obtained  with  non-specific  lipoidal  sub- 
stances. Although  we  are,  at  the  present  writing,  still  in  the  dark  as 
to  whether  the  syphilitic  antigen  depends  for  its  properties  upon 
the  lipoidal  nature  of  the  extracts  or  upon  the  size  and  disposition  of 
the  particles  present  in  the  extracts,  we  can  still  assert  that  the  test  is 
reliable  and,  with  care  in  execution  and  interpretation,  of  enormous 
value  in  the  diagnosis  of  syphilis.  However,  it  is  necessary  to  recog- 
nize that  it  is  surely  not  a  specific  antigen-antibody  reaction. 

The  antigens  most  commonly  in  use  today  are  prepared  as  follows : 

1.  Beef  heart  or  guinea-pig  heart  muscle  is  finely  chopped  up  and 
extracted  in  five  times  its  volume  of  absolute  alcohol.     This  mixture 
is  kept  5  to  7  days  in  the  incubator,  being  frequently  shaken.     It  is 
then  filtered  and  titrated.     Human  heart  muscle  may  also  be  used. 

2.  Noguchi's  Acetone  Insoluble  Lipoid  Antigen.     Fresh  spleen  is 
macerated  and  extracted  for  5  to  7  days  in  the  incubator  in  five  times 
its  volume  of  absolute  alcohol,  being  frequently  shaken.     It  is  then 
filtered  and  evaporated  to  dryness  with  the  aid  of  a  fan.     The  sticky 
residue  is  taken  up  in  a  small  quantity  of  ether  and  this  ether  solution 
poured  into  four  times  its  volume  of  C.  P.  acetone.     The  floccular 
precipitate   which   forms   is   collected   and   can   be   preserved   under 
acetone.    About  0.2  gram  of  this  paste  is  dissolved  in  5  c.c.  of  ether. 
This  is  shaken  up  with  100  c.c.  of  salt  solution  until  the  ether  is 
evaporated.    The  resulting  antigen  is  titrated. 

.  3.  Chloresterinized  Antigen.  According  to  the  researches  of  Sachs 
and  Rondoni,  Browning  and  Cruikshank,  and  Walker  and  Swift, 
antigen  can  be  made  more  delicate  by  the  addition  of  cholesterin. 
Walker  and  Swift  recommend  that  an  alcoholic  extract  of  human  or 
guinea-pig  heart  be  made  up  to  a  concentration  of  0.4  per  cent  of 
cholesterin. 

A  large  number  of  other  antigens  might  be  mentioned,  but  we 
think  that  the  three  mentioned  above  represent  the  most  important, 
and  in  principle  all  of  those  at  present  in  common  use. 

Before  an  antigen  can  be  used  for  the  actual  test,  it  is  necessary  to 
determine  the  quantity  which  will  furnish  a  valid  result.  The  sub- 
stances which  are  used  as  antigens  often  have  the  power,  if  used  in  too 
large  quantity,  of  themselves  binding  complement.  It  is  necessary, 
therefore,  to  determine  the  largest  quantity  of  each  given  antigen 
which  may  be  used  without  exerting  an  anti-complementary  action, 
i.e.,  which  will  not  inhibit  in  the  presence  of  normal  serum  but  which 
will  at  the  same  time  inhibit  hemolysis  when  syphilitic  serum  is  used. 


THE  TECHNIQUE  OF  SERUM   REACTIONS  319 

This  is  done  by  mixing  graded  quantities  of  the  antigen  with  a  con- 
stant quantity  of  complement  (0.1  c.c.  of  fresh  guinea-pig  serum),  in 
duplicate  sets,  adding  to  each  tube  of  one  set  0.2  c.c.  of  a  normal 
serum,  and  to  the  other  0.2  c.c.  of  a  known  syphilitic  serum.  These 
substances  are  allowed  to  remain  together  for  one  hour  and  then  red 
blood  corpuscles  and  inactivated  hemolytic  serum  are  added.  The 
quantity  which  has  given  complete  inhibition  with  the  syphilitic  serum, 
but  absolutely  no  inhibition  with  normal  serum,  is  the  one  to  be 
employed  in  subsequent  reactions.  Before  actual  use,  it  is  convenient 
to  make  a  dilution  of  antigen  in  salt  solution  in  such  a  way  that  1  c.c. 
shall  contain  the  amount  required.  Thus  if  0.05  c.c.  is  wanted,  mix 
0.5  c.c.  with  9.5  c.c.  salt  solution.  Then  1  c.c.  of  this  can  be  added  to 
each  tube  in  the  test. 

II.  The  Hemolytic  Serum. — The  hemolytic  amboceptor,  for  the 
reaction,  is  obtained  by  injecting  into  rabbits  the  washed  red  blood 
corpuscles  of  a  sheep.  A  5  per  cent  emulsion  of  the  corpuscles  is  made 
and  of  this  5  c.c.,  10  c.c.,  15  c.c.,  etc.,  are  injected  at  intervals  of  five 
or  six  days.  Three  or  four  graded  injections  of  this  kind  are  usually 
sufficient  to  furnish  a  serum  of  adequate  hemolytic  power.  The  injec- 
tions may  be  made  intraperitoneally  or  intravenously.  About  nine  or 
ten  days  after  the  last  injection  of  corpuscles,  the  rabbit  is  bled  from 
the  carotid  artery  and  the  serum  obtained  by  pipetting  it  from  the 
clot. 

It  is  best  to  have  a  hemolytic  serum  of  high  potency  in  order  that 
the  quantities  used  for  the  reaction  may  be  as  small  as  possible.  This 
is  desirable  because  of  the  fact  that  the  serum  may  contain  small 
amounts  of  precipitins  for  sheep's  serum,  due  to  insufficient  washing 
of  the  corpuscles  employed  in  the  immunization. 

It  is  necessary  to  carefully  titrate  the  hemolytic  serum.  For  the 
actual  reaction  most  observers  make  use  of  two  hemolytic  units.  A 
hemolytic  unit  is  the  quantity  of  inactivated  immune  serum  which,  in 
the  presence  of  complement,  suffices  to  cause  complete  hemolysis  in 
1  c.c.  of  a  5  per  cent  emulsion  of  washed  blood  corpuscles.  It  is  the 
custom  in  most  laboratories  today  to  halve  all  the  quantities,  using 
0.5  c.c.  of  the  suspension  instead  of  1.0  c.c.  and  other  ingredients 
accordingly.  Noguchi 15  has  pointed  out  very  clearly  the  dangers  of 
not  delicately  adjusting  the  quantity  of  amboceptor  used  in  the  reac- 


NogucJii,  Poc.  Soc.  for  Exper.  Biol.  and  Med.,  VI,  3,  1909. 


320 


INFECTION  AND  IMMUNITY 


tion.  He  calls  attention  to  the  experiments  of  Morgenroth  and  Sachs 10 
who  have  shown  that  the  relationship  between  complement  and  ambo- 
ceptor  necessary  for  hemolytic  reactions  is  one  of  inverse  proportions. 
In  their  own  words,  "in  the  presence  of  larger  quantities  of  ambo- 
ceptor,  smaller  quantities  of  complement  suffice,"  and  vice  versa. 
Noguchi,  in  his  work,  has  found  that,  while  in  the  presence  of  one 
unit  of  amboceptor,  0.1  c.c.  of  guinea-pig's  complement  is  required  to 
produce  hemolysis,  by  using  four,  eight,  and  twenty  units  of  ambo- 
ceptor, complete  hemolysis  is  obtainable  with  one-third,  one-fifth,  and 
one-tenth  of  the  0.1  c.c.  of  complement,  respectively.  For  this  reason 
an  excess  of  amboceptor  might  result  in  complete  hemolysis  in  a  test, 
if  a  small  fraction  of  the  complement  were  left  unfixed  by  the  syphil- 
itic antibody.  Another  result  of  an  excess  of  amboceptor  would  con- 
sist in  a  partial  dissociation  of  the  complement  from  its  combination 
with  the  antigen-antibody  compound.  As  Noguchi  puts  it,  "  a  quantity 
of  syphilitic  antibody  just  sufficient  to  fix  0.1  c.c.  of  the  complement 
against  two  units  of  the  amboceptor  is  no  longer  efficient  in  holding 
back  the  complement  from  partial  liberation  against  the  influence 
exerted  by  more  than  four  units  of  the  amboceptor." 

From  these  considerations  it  follows  that  the  serum  from  rabbits 
immunized  against  sheep  corpuscles  must,  in  each  case,  be  titrated  in 
order  to  determine  the  hemolytic  unit.  For  this  purpose  a  number  of 
mixtures  are  made  in  test  tubes,  containing  each  0.1  c.c.  of  complement 
(fresh  guinea-pig  serum),  1  c.c.  of  a  5  per  cent  emulsion  of  sheep 's 
corpuscles,  and  diminishing  quantities  of  the  inactivated  hemolytic 
serum,  thus: 


1  c.c. 

.01  c  .c  .  =  complete  hemolysis 

.1  c.c.  of 
complement 
fresh 
guinea-pig 

+  < 

of  5  per 
cent 
emul- 
sion 
sheep's 

'  + 

Inac- 
tivated 
hemo- 
lytic 

.  009  c  .c  .  =  complete  hemolysis 
.  005  c  .c  .  =  complete  hemolysis 
.003  c.c.  =  complete  hemolysis 
.  001  c  .c  .  =  complete  hemolysis 
.0009  c.c.=  partial  hemolysis 

serum.       , 

corpus- 

serum. 

.0005  c.c.=no  hemolysis 

cles,     i 

.0003  c.c.=no  hemolysis  17 

In  the  given  case,  0.001  c.c.  of  the  serum  represents  one  unit,  and 
0.002  c.c.,  two  units,  is  the  quantity  to  be  used  for  each  test. 

16  Morgenroth  und  Sachs,  in   Ehrlich's  ' '  Gesammelte  Arbeiten,"   etc.,  Berlin, 
1904. 

17  In  each  tube  the  volume  of  the  mixture  should  be  made  up  to  5  c.c.  with 
0.85  per  cent  salt  solution. 


THE  TECHNIQUE   OF  SERUM   REACTIONS  321 

III.  The    Complement. — The    complement   used   in   Wassermann 
reaction  is  fresh  guinea-pig  serum.     This  may  be  obtained  in  one  of 
the  following  ways :   A  guinea-pig  may  be  killed  by  an  incision  in  the 
throat  and  the  blood  allowed  to  flow  into  a  large  Petri  dish.     This  is 
set  away  in  the  ice  chest  until  clear  beads  of  serum  have  formed 
upon  the   surface,   and  these   are  then  carefully  removed  with  a 
pipette. 

It  is  more  economical  to  puncture  the  heart  of  large  guinea-pigs 
with  a  needle  attached  to  a  syringe  and  withdraw  5  or  6  c.c.  of  blood 
without  killing  the  animal.  This  can  be  transferred  to  a  centrifuge 
tube  and  the  serum  obtained  by  centrifugation  after  clotting.  Serum 
used  as  complement  in  the  Wassermann  reaction  must  be  titrated  each 
day  before  reactions  are  done.  This  is  done  by  putting  into  a  series 
of  tubes  1.0  c.c.  (or  if  half  quantities  are  used,  as  with  us,  0.5  c.c.) 
of  the  cell  suspension  sensitized  with  2  units  of  amboceptor,  and 
adding  to  these  tubes  varying  quantities  of  guinea-pig  serum.  The 
guinea-pig  serum  is  best  diluted  1 :10  in  salt  solution,  and  quantities 
ranging  from  0.05  to  0.35  c.c.  are  added  to  the  tubes.  The  unit  is  the 
amount  in  the  tube  which  shows  complete  hemolysis  at  the  end  of  an 
hour.  The  reactions  are  usually  complete  in  about  30  minutes.  Two 
units  of  the  complement  are  used  in  the  ordinary  test.  The  titration 
of  the  complement  is  one  of  the  most  important  steps  in  accurate 
work. 

IV.  The  Sheep  Corpuscles. — The  sheep  corpuscles  for  the  actual 
reaction  are  obtained  by  receiving  the  blood  in  a  small  flask  containing 
a  sterile  solution  of  a  0.5  per  cent  sodium  citrate  and  0.85  per  cent 
sodium  chloride,  or  into  one  containing  glass  beads  or  short  pieces  of 
glass  tubing.    In  the  former  case,  the  citrate  solution  prevents  clotting 
and  the  corpuscles  may  be  washed  free  from  the  citrate  solution  and 
emulsified  in  salt  solution  before  use  in  the  test.    In  the  latter  case,  it 
is  necessary  to  shake  the  blood  in  the  flask  immediately  after  taking, 
and  to  continue  the  shaking  motion  for  about  ten  minutes.     The  cor- 
puscles are  washed  free  from  serum  by  at  least  3  washings  in  salt 
solution.     A  5  per  cent  suspension  of  the  corpuscles  is  employed  for 
the  test,  made  by  measuring  the  bulk  of  centrifugalized  corpuscles  and 
adding  nineteen  parts  of  sterile  salt  solution. 

V.  The  Serum  to  be  Tested  for  Syphilitic  Antibody. — The  serum  of 
the  patient  is  best  obtained  in  the  same  way  that  blood  is  obtained  for 
blood  cultures.     After .  surgical  precautions,  a  needle  is  plunged  into 
the  median  basilic  vein  and  3  or  4  c.c.  of  blood  are  removed.    Before 


322 


INFECTION   AND   IMMUNITY 


use  for  the  test,  the  patient's  serum  must  be  inactivated  by  heating  in 
a  water  bath  to  56°  C.  for  twenty  minutes  to  half  an  hour. 

THE  TEST. — The  actual  test  for  antibody  in  a  suspected  serum  is 
carried  out  in  the  following  way:  In  a  test-tube  of  suitable  size,  2 
units  of  the  complement,  0.2  c.c.  of  the  inactivated  suspected  serum, 
and  the  antigen,  in  quantity  determined  by  titratioii,  are  mixed,  and 
the  total  volume  brought  up  to  3  c.c.  with  normal  salt  solution.  This 
mixture  is  thoroughly  shaken,  and  placed  for  one  hour  in  a  water 
bath  or  in  the  incubator  at  37.5°  C.  Recently  it  has  been  found  that 
more  delicate  results  are  obtained  when  the  fixation  is  allowed  to  take 
place  in  the  refrigerator  for  three  or  four  hours — the  so-called  ' '  ice-box 
method."  At  the  end  of  this  preliminary  incubation  1  c.c.  of  a 
5  per  cent  emulsion  of  sheep 's  corpuscles,  and  two  units  of  hemolytic 
amboceptor,  determined  by  a  titration  of  the  inactivated  hemolytic 
rabbit  serum,  as  described  above,  are  added.  This  mixture  is  again 
placed  at  37.5°  C.  for  one  to  two  hours.  If  the  antibody  is  present  in 
the  suspected  serum,  no  hemolysis  takes  place.  If  absent,  hemolysis  is 
complete. 

In  our  own  work  all  tests  are  done  in  half  the  quantities  of  the 
original  Wassermann.  Hence  only  0.1  c.c.  of  the  patient's  serum,  and 
the  antigen  and  complement  as  determined  in  titrations  with  0.5  c.c. 
of  the  cells  are  mixed  in  a  total  volume  of  1.5  c.c.  At  the  end  of  the 
preliminary  incubation,  0.5  c.c.  of  cells  previously  sensitized  with  2 
units  of  amboceptor  are  added. 

No  test  is  of  use  unless  suitable  controls  are  made.  The  controls 
set  up  should  be  as  follows : 

Control  1.  For  each  serum  tested  the  mixture  described  above, 
omitting  antigen. 

Controls  2  and  3.  The  mixture  made  as  in  the  test  but  with 
known  syphilitic  serum  (2)  with  and  (3)  without  antigen. 

Controls  4  and  5.  The  mixture  made  as  in  the  test,  but  with 
normal  serum  (4)  with  and  (5)  without  antigen. 

Controls  6  and  7.  The  hemolytic  system,  complement,  blood  cells 
and  amboceptor,  set  up  in  order  to  show  that  the  system  is  in  working 
order  (6)  with  and  (7)  without  antigen.  It  is  convenient  to  set  the 
tubes  in  two  rows  in  a  rack,  the  front  row  containing  antigen,  the 
back  row  containing  the  same  mixture  without  antigen. 

In*  a  positive  test,  the  test  itself,  and  Control  2,  alone,  should  show 
inhibited  hemolysis.  The  other  tubes  should  show  complete  solution 
of  the  hemoglobin.  (See  scheme,  p.  324.) 


THE  TECHNIQUE  OF  SERUM   REACTIONS  323 

Modifications  of  the  Wassennann  Test.— Since  the  original  for- 
mulation of  the  Wassermann  reaction  a  great  many  modifications  have 
been  suggested  by  various  workers,  some  of  them  being  radical  changes 
involving  the  altering  of  the  hemolytic  system ;  others,  however,  merely 
adding  precautions  here  and  there  to  increase  the  delicacy  of  the 
reaction.  The  literature  on  this  subject  is  too  voluminous  to  be  com- 
pletely covered.  We  indicate,  therefore,  some  of  the  most  important 
changes  from  the  original  that  have  been  found  valuable,  and  give  in 
greater  detail  the  methods  as  at  present  in  use  in  our  own  laboratory. 

Bauer  has  called  attention  that  human  serum  contains  a  certain  amount  of 
natural  hemolysin  for  sheep  corpuscles.  In  his  original  modification,  there- 
fore, he  does  not  use  hemolytic  rabbit  serum  as  amboceptor.  His  modification 
as  a  whole  cannot  be  accepted  for  general  use  because  human  sera  do  not 
contain  a  uniform  amount  of  hemolysin  for  sheep  cells,  and  some  contain 
none  whatever.  However,  the  presence  of  natural  amboceptor,  so-called,  in 
human  sera  is  taken  account  of  by  many  workers,  and  it  is  important  to 
recognize  this,  since  naturally  it  adds  to  the  amboceptor  added  with  sensitized 
cells  and  leads  to  a  lack  of  uniformity  in  the  dosage  of  amboceptor  in 
individual  tubes  if  included. 

Noguchi  has  worked  out  a  test  in  which  the  difficulties  presented  by  the 
presence  of  normal  sheep  amboceptor  are  eliminated,  in  that  he  uses  an 
antihuman  hemolytic  serum  and  human  cells  as  the  hemolytic  system.  It 
enables  him  also  to  use  the  cells  of  the  patient  or  of  any  other  human  being, 
thus  eliminating  the  necessity  of  getting  fresh  sheep  cells.  His  tes'ts  are 
set  up  as  follows: 

Tube  1.  1  drop  patient's  serum  -{-  complement  (0.1  c.c.  of  40  per  cent  guinea-pig 

serum)  -|-  antigen. 

Tube  2.  1  drop  patient's  serum  -|-  complement.     (No  antigen.) 

Tube  3.  1  drop  known  syphilitic  serum  -|-  complement  -|-  antigen. 

Tube  4.  1  drop  known  syphilitic  serum  -|-  complement.      (No  antigen.) 

Tube  5.  1  drop  known  normal  serum  -(-  complement  -f-  antigen. 

Tube  6.  1  drop  known  normal  serum  -j-  complement.      (No  antigen.) 

Tube  7.  Complement  alone   (for  hemolytic  system  control). 

To  each  tube  then  add  1.0  c.c.  of  the  one  per  cent  emulsion  of  human 
corpuscles.  Shake  mixtures  thoroughly  and  incubate  or  place  in  water  bath 
at  38-40°  C.  for  one  hour.  Then  add  to  each  tube  2  units  of  antihuman 
amboceptor  (serum  of  rabbit  immunized  with  human  cells)  and  replace  in 
water  bath  for  one  hour.  At  the  end  of  this  time  in  a  positive  test  there 
will  be  no  hemolysis  in  Tubes  1  and  3  while  all  the  other  tubes  will  show 
hemolys'is. 


324 


INFECTION  AND   IMMUNITY 


The  method  of  Noguchi  is  still  used  by  a  few  investigators,  but  is 
not  at  present  in  common  use,  though  we  have  no  doubt  that  if  sys- 
tematically followed  it  would  develop  as  quite  satisfactory. 

The  tests  are  done  in  our  own  laboratory  with  the  original  sheep 
cell — antisheep  serum  hemolytic  system.  They  are  done  in  half 
quantities,  titrations  being  made  with  0.5  c.c.  of  a  5  per  cent  emulsion 
of  washed  sheep  cells.  Each  day  the  complement  (fresh  guinea-pig 
diluted  1:10)  is  titrated  with  cells  sensitized  with  2  units  of  stock 
amboceptor.  Fresh  amboceptors  are  titrated  from  time  to  time  so 
that  a  reasonable  constancy  is  obtained.  The  hemolytic  system  is  kept 
as  constant  as  possible  from  day  to  day.  The  unit  (minimal  hemolytic 
amount)  of  a  new  specimen  of  amboceptor  is  determined  by  titrating 

SCHEME  FOR  WASSERMANN  TEST. 

ADAPTED   TO   ORIGINAL  WASSERMANN   SYSTEM   AFTER   SCHEME   OF  NOGUCHI. 


Test  without  Serum 

• 

Test  with  Unknown 
Serum. 

Test  with  Known 
Positive  Syphilic 

Test  with  Known 
Negative  Normal 

to  Control  Efficiency 
of  Hemolytic 

Serum. 

Serum. 

System. 

d 

§ 

Serum  .2  c.c. 

13 

Serum  .2  c.c. 

Serum  .2  c.c. 

^  •£ 

. 

CO 

. 

si 

O   Complement 

oJ 

O    Complement 

O   Complement 

O   Complement 

1  j 

.1  c.c. 

'o 

.1  c.c. 

.1  c.c. 

.  1  c.c 

PQ| 

Salt  sol.  3.  c.c. 

3 

o 

Salt  sol.  3.  c.c. 

Salt  sol.  3.  c.c. 

Salt  sol.  3.  c.c. 

2. 

P 

4. 

6. 

8. 

Serum  .2  c.c. 

Serum  .2  c.c. 

Serum  .2  c.c. 

-f- 

d 

-)- 

-)- 

§• 

O   Complement 

CO 

O   Complement 

O   Complement 

O   Complement 

w  a 

.1  c.c. 

.1  c.c. 

.1  c.c. 

.1  c.c. 

Ix 

Antigen 

1 

Antigen 

Antigen 

Antigen 

fa    ^ 

(required  amount 

13 

. 

. 

\/ 

in  1  c.c.  salt  sol.). 

1 

' 

' 

/\ 

Salt  sol.  2.  c.c. 

P 

Salt  sol.  2.  c.c. 

Salt  sol.  2.  c.c. 

Salt  sol.  2.  c.c. 

1. 

3. 

5. 

7. 

O  =test  tube. 

Place  in  water  bath  at  40°  C.  for  one  hour,  then  add  to  all  tubes  red  blood  cells 
and  amboceptor.  These  are  previously  mixed  so  that  2  c.c.  contains  the  equivalents 
of  1  c.c.  of  a  5  per  cent  emulsion  of  sheep  corpuscles  and  2  units  of  amboceptor. 
Again  expose  to  40°  C.  If  the  serum  tested  is  positive,  tubes  1  and  3  should  show 
no  hemolysis,  all  the  other  tubes  showing  complete  hemolysis  in  one  hour. 


THE  TECHNIQUE  OF  SERUM  REACTIONS  325 

it  on  a  number  of  successive  days  with  0.5  c.c.  of  5  per  cent  cells  and 
0.5  c.c.  of  10  per  cent  guinea-pig  sera,  readings  being  made  at  the  end 
of  a  half  hour.  When  complement  is  then  subsequently  titrated  with 
2  such  amboceptor  units  and  0.5  c.c.  of  5  per  cent  red  cells,  it  is  usually 
found  that  the  minimal  hemolytic  dose  of  complement  lies  between  0.2 
and  0.25  c.c.,  and  in  the  actual  tests  twice  this  minimal  hemolytic  dose 
of  complement  is  used  with  the  2  units  of  amboceptor.  The  daily 
titration  of  complement  frequently  shows  marked  variations  even 
though  the  complement  is  obtained  from  a  number  of  guinea-pigs.  By 
always  using  twice  the  amount  which  just  lakes  with  2  units  of  ambo- 
ceptor, one  keeps  the  actual  amount  of  complement  as  nearly  as  pos- 
sible constant. 

Obviously  one  can  titrate  the  strength  of  the  reaction  by  varying 
the  amount  of  any  one  of  the  three  ingredients  which  primarily  enter 
into  it  —  the  antigen,  the  patient's  serum,  or  the  complement,  and  all 
three  of  these  systems  have  been  proposed  and  used  with  success  by 
different  workers.  The  method  which  is  most  commonly  used  is  that 
of  Citron  and  consists  of  using  diminishing  quantities  of  both  antigen 
and  patient's  serum.  Citron  in  addition  to  the  main  tubes  uses  one 
additional  tube  containing  one-half  the  amount  of  patient's  serum  and 
one-half  the  amount  of  antigen  used  in  the  main  tube,  and  he  expresses 
his  results  as  follows: 


If  both  tubes  show  complete  inhibition  .................  .  ........  +  + 

If  the  main  tube  shows  complete  inhibition  and  the  half  dose  tube 

almost  complete  ...........................................       +  +  + 

If  the  main  tube  shows  complete  inhibition  and  the  second  tube 

faint  inhibition   ...................  ........................  -f-  ~h 

If  the  main  tube  shows  incomplete  inhibition  and  the  second  tube 

none  or  little   .............................  ................  + 

If  the  main  tube  shows  very  faint  inhibition  and  the  second  tube 

shows  none   ...............................................  ± 

"-!-  +  +  +  ,"  "  +  +  +  ,"  and  "  +  +  "  are  regarded  as  conclusive  results; 
"  +  "  as  a  probable  positive;  "  ±  "  as  suspicious  merely. 

The  Determination  of  Antigen  by  Complement  Fixation.  —  The 

principles  underlying  the  preceding  tests  for  the  determination  of 
suspected  antibodies  may  be  equally  applied  to  the  determination  of 
suspected  antigen.  In  the  former  case  it  was  necessary  to  bring  the 
serum  to  be  tested  into  contact  with  the  antigen  specific  for  the 
suspected  antibody,  in  the  presence  of  complement,  and  at  a  suitable 
temperature.  At  the  end  of  an  hour  the  mixture  was  tested  for  free 


326  INFECTION   AND  IMMUNITY 

complement  by  the  addition  of  hemolytic  amboceptor  and  red  blood 
cells.  In  testing  for  antigen,  the  procedure  is  reversed,  in  that  the 
serum  or  other  substance  (bacterial  extract)  to  be  tested  is  brought 
into  contact  with  an  antibody  specific  for  the  antigen,  in  the  presence 
of  complement;  and  at  the  end  of  an  hour  at  suitable  temperature, 
free  complement  is  again  determined  by  hemolytic  reaction  as  before. 

When  dealing  with  bacterial  antigen,  it  is  necessary,  therefore,  to 
prepare  a  highly  potent  immune  serum  against  the  bacteria  which  con- 
tain the  specific  antigen  which  is  sought. 

Thus  in  testing  for  typhoid-bacillus  antigen  in  the  serum  of  a 
patient,  the  substances  required  are  as  follows : 

1.  Complement:    obtained   from   fresh   guinea-pig   serum.      It   is 
best  to  titrate  the  complement  when  possible,  using  for  the  test  double 
the  quantity  necessary  to  produce  complete  hemolysis  of  1  c.c.  of  a 
five  per  cent  emulsion  of  blood  cells,  in  the  presence  of  two  units  of 
amboceptor. 

2.  Hemolytic  amboceptor:    rabbit  serum  hemolytic  for  sheep  cor- 
puscles.    Inactivated  and  titrated  as  for  Wassermann  test. 

3.  A  five-per-cent  emulsion  of  sheep  corpuscles  in  salt  solution, 
prepared  as  for  Wassermann  test. 

4.  A  highly  potent  typhoid  antiserum  obtained  from  an  immunized 
rabbit.    In  this  case  the  smallest  quantity  of  the  immune  serum  which 
will  cause  the  fixation  of  complement  in  the  presence  of  an  emulsion 
or  extract  of  known  typhoid  bacilli  is  determined  by  experiment. 

It  is  best  to  rub  up  the  centrifugalized  bacteria  with  dry  salt, 
adding  distilled  water  to  isotonicity.  When  the  extract  is  made,  its 
anticomplementary  dose  is  determined  and  the  minimum  quantity 
which  in  the  presence  of  known  antityphoid  serum  will  fix  complement. 
These  preliminary  titrations  are  analogous  to  those  described  as  pre- 
liminary to  the  Wassermann  test.  When  these  quantities  have  been 
determined,  an  amount  of  the  bacillary  extract  (about  1-3  or  1-4  of 
the  anticomplementary  dose)  is  chosen  for  the  actual  tests.  It  is  well, 
also,  in  such  tests  to  determine  the  amount  which  will  fix  complement 
in  the  presence  of  a  known  normal  serum,  since  occasional  presence  of 
antibodies  against  some  bacteria  in  normal  serum  may  otherwise  con- 
fuse the  test.  The  quantities  of  antigen  and  complement  must  then  be 
chosen  for  the  test  in  such  proportions  that  no  fixation  will  occur  with 
normal  serum. 

5.  Serum   from   the   patient,    inactivated   at   56°    C.   for   twenty 
minutes. 


THE  TECHNIQUE  OF  SERUM   REACTIONS  327 

In  the  actual  test  a  -series  of  tubes  are  prepared  each  of  which  con- 
tains : 

1.  Complement,  the  determined  quantity. 

2.  Antiserum,  the  determined  quantity. 

3.  Diminishing  quantities  of  the  inactivated  serum  to  be  tested  for 
antigen  beginning  with  1  c.c. 

Salt  solution  is  added  for  dilution  to  3  c.c. 

These  substances  are  left  together  at  37°  to  40°  C.  for  one  hour  and 
then  the  required  quantities  of  amboceptor  and  red  cells  are  added. 
The  reaction  is  controlled  by  tubes  containing  the  same  ingredients 
without  the  typhoid  antiserum.  In  a  positive  test  there  will  be  no 
hemolysis  in  the  tubes  containing  the  patient's  serum. 

Protein  Differentiation  by  Complement  Fixation. — That  the  tech- 
nique of  complement  fixation  was  applicable  to  the  determination  of 
specific  protein  antigen — such  as  human  or  animal  blood — was  shown 
by  Gengou  18  in  1902.  The  principles  worked  out  by  him  have  been 
practically  applied  by  Neisser  and  Sachs  19  and  others  to  the  forensic 
differentiation  of  animal  proteins  and  these  tests  are  said  to  be  more 
delicate  and  reliable  than  precipitation  tests  made  for  the  same  pur- 
pose. 

The  substances  necessary  for  the  reaction  are  as  follows : 

1.  Complement,  titrated  as  above. 

2.  Hemolytic  amboceptor  as  above. 

3.  A  five-per-cent  emulsion  of  sheep  corpuscles  as  above. 

4.  Specific  antiserum. 

This  is  obtained  from  a  rabbit  immunized  with  the  protein  for 
which  the  test  is  to  be  made ;  viz. :  human  or  animal  blood  serum. 
This  must  be  titrated.  In  order  to  do  this,  diminishing  quantities  of 
the  antiserum  are  mixed  in  a  series  of  tubes  with  the  determined 
quantity  of  complement,  and  the  antigen  which  is  to  be  tested  for,  i.e., 
the  homologous  serum  with  which  the  antiserum  has  been  produced. 
Since  the  test  should  be  sufficiently  delicate  to  determine  0.0001  c.c. 
of  the  antigen,  this  quantity  is  added  to  each  tube.  The  actual  titra- 
tion  is  as  follows : 20 

™  Gengou,  Ann.  de  1'Inst.  Pasteur,  1902.  % 

w  Neisser  imd  Sachs,  Berl.  klin.  Woch.,   1905   and  1906,     See  also  Citron,  in 
Kraus  and  Levaditi  ' '  Handbuch/ '  etc. 
-(}  Citron,  loe.  cit. 


328  INFECTION  AND  IMMUNITY 

1.  Antiserum,  0.1    +  homologous  serum  .0001  +  complement 

2.  Antiserum,  0.05  +  homologous  serum  .0001  +  complement 

3.  Antiserum,   0.02  +  homologous  serum  .0001  +  complement 

4.  Antiserum,   0.01  +  homologous  serum  .0001  +  complement 
(Antiserum  and  homologous  serum  each  brought  to  1  c.c.  volume 

with  salt  solution.) 

These  tubes  are  incubated  for  one  hour  and  hemolytic  amboceptor 
and  red  blood  cells  are  added.  The  smallest  quantity  of  antiserum 
which  has  completely  inhibited  hemolysis  is  the  "unit,"  and  one  and  a 
half  to  two  times  this  quantity  is  used  for  the  test. 

5.  A  solution  of  the  blood  spot  or  other  material  to  be  tested  pre- 
pared as  for  precipitin  test.  (See  page  306.) 

For  the  actual  test  the  following  mixtures  are  made  in  a  series  of 
tubes,  each  of  which  contains : 

1.  Complement ) 

I  quantity  determined  by  titration. 

2.  Antiserum     J 

3.  Diminishing  quantities  of  the  substance  in  which  the  antigen  is  sus- 
pected, ranging  from  0.1  c.c.  downward  to  0.0001  c.c. 

Salt  solution  is  added  as  a  diluent  up  to  3  c.c.  and  the  tubes  are 
placed  in  the  incubator  or  water-bath  at  37.5°  to  40°  C.  At  the  end 
of  this  time  red  blood  cells  and  amboceptor  are  added  as  before. 

The  tubes  are  controlled  by  a  series  containing  all  the  above  ingre- 
dients except  the  antiserum. 

SACHS-GEORGI  REACTION  FOR  SYPHILIS*   l 

(Direct  Precipitation) 

PREPARATION  OF  EXTRACT. — A  beef  heart  is  freed  from  fat  and 
endocardium,  cut  up  finely  and  ground  in  a  mortar.  It  is  then  shaken 
with  5  volumes  of  95  per  cent  alcohol  and  a  few  glass  beads  in  a 
shaking  machine  for  5  hours,  allowed  to  stand  at  room  temperature 
over  night,  filtered  through  ordinary  filter  paper  next  morning,  then 
placed  in  the  ice-box  for  at  least  two  days,  when  it  is  again  filtered 
through  ordinary  filter  paper,  and  is  now  ready  for  use.  It  must 
first  be  titrated  against  a  standard  extract  on  a  number  of  sera  to 
determine  the  optimum  dilution  and  cholestermization.  For  this,  the 
alcoholic  extract  is  diluted  with  1,  2,  and  3  parts  of  alcohol,  and  to 
fractions  of  each  of  these  dilutions,  0.3,  0.45,  0.6  and  0.75  per  cent  of 

*  For  this  description  we  are  indebted  to  our  associate,  Dr.  Fred  'k.  Parker, 
Jr.,  who  has  given  particular  attention  to  this  reaction. 


THE   TECHNIQUE  OF  SERUM   REACTIONS  329 

a  1  per  cent  alcoholic  solution  of  cholesterin  is  added.  These  various 
portions  are  then  diluted  with  5  parts  of  saline  as  described  below,  and 
set  up  against  a  standard  extract.  At  least  two  such  extracts  should  be 
used  in  each  test. 

EXTRACT  DILUTION. — The  alcoholic  cholesterinized  extract  is  diluted 
with  5  parts  of  saline  as  follows:  The  required  amount  of  extract  is 
placed  in  an  Erlenmeyer  flask ;  to  it  is  rapidly  added  from  a  burette 
an  equal  volume  of  saline.  It  is  shaken  gently  and  allowed  to  stand 
10  minutes,  then  the  remaining  4  volumes  of  saline  are  rapidly  run 
in.  It  is  again  shaken  and  is  ready  for  use. 

SERUM. — Serum  should  be  as  fresh  as  possible.  Three  or  four  days 
is  not  too  old.  A  slight  degree  of  hemolysis  does  not  interfere.  Before 
use  in  the  test,  it  is  heated  for  y2  hour  at  55°  to  56°  C.  and  should 
not  be  used  sooner  than  3  hours  after  heating.  Sachs  and  Georgi21 
recommend  that  spinal  fluids  should  be  used  undiluted  in  amounts  of 
1  c.c.  and  0.5  c.c. 

Saline. — 0.85  per  cent  sodium  chlorid  in  distilled  water.  Should 
be  sterile  and  as  fresh  as  possible. 

Test. — 0.1  c.c.  serum  is  diluted  with  0.9  c.c.  saline  and  to  this  is 
added  0.5  c.c.  extract  dilution.  On  each  serum  a  control  should  be 
set  up  consisting  of  0.1  c.c.  serum  -f-  0.9  c.c.  saline  +  0.5  c.c.  of  95  per 
cent  alcohol,  diluted  1 :6  with  saline.  Each  extract  dilution  should  be 
controlled  by  a  tube  containing  0.5  c.c.  extract  dilution  +  1  c.c.  saline. 

The  tubes  are  thoroughly  shaken  and  placed  in  the  incubator  at 
37.5°  C.  for  20  hours.  A  preliminary  reading  may  now  be  made; 
then  the  tubes  are  placed  at  14°  to  18°  C.,  or  in  the  ice-box  for  20 
hours,  and  the  final  and  decisive  readings  are  taken. 

The  reactions  present  appearances  similar  to  macroscopic  bacterial 
agglutination  or  precipitin  reactions,  and  are  read  with  the  naked 
eye,  the  positives  showing  varying  amounts  of  precipitates  and  the 
negatives  remaining  opalescent  as  at  the  beginning  of  the  test.  Sus- 
picious tests  are  centrifuged  at  moderate  speed  for  a  few  minutes,  and 
are  proved  positive  or  negative  by  the  fact  that  in  the  positives  after 
centrifuging  a  few  definite  white  compact  flocculi  can  be  shaken  from 
the  bottom  of  the  tube,  whereas  the  negatives,  at  most,  show  a  slight 
grayish  sediment  which  disperses  on  shaking.  The  serum  controls 
should  remain  clear.  If  a  precipitate  does  occur,  the  serum  is  unsuit- 
able and  another  specimen  must  be  obtained.  The  extract  controls 
should  remain  diffusely  opalescent,  and  should  show  no  precipitate. 

21  Sachs-Georgi,  Med.  Klinik,  No.  33,  1918,  805;  Parker  and  Haigh,  un- 
published. 


CHAPTER  XVII 

PHAGOCYTOSIS 

THE  studies  on  immunity  which  we  have  outlined  in  the  preceding 
sections  have  dealt  entirely  with  the  phenomena  occurring  in  the 
reaction  between  bacteria  or  bacterial  products  and  the  body  fluids. 
These  studies,  we  have  seen,  have  formed  the  basis  of  a  theoretical 
conception  of  immunity  formulated  chiefly  by  the  German  school  of 
bacteriologists  under  the  leadership  of  Ehrlich,  Pfeiffer,  Kruse,  and 
others.  Parallel  with  these  developments,  however,  investigations  on 
immunity  have  been  carried  on  which  have  brought  to  light  many 
important  facts  concerning  the  participation  of  the  cellular  elements 
of  the  body  in  its  resistance  to  infectious  germs. 

The  inspiration  for  this  work  and  the  greater  part  of  the  theoretical 
considerations  which  have  been  based  upon  it,  have  emanated  from 
Metchnikon3 1  and  his  numerous  pupils  at  the  Pasteur  Institute  in 
Paris.  The  phenomenon  which  these  observers  have  studied  in  great 
detail  and  upon  the  occurrence  of  which  they  have  based  their  con- 
ceptions of  immunity,  is  known  as  phagocytosis. 

It  is  well  known  that  among  the  lowest  unicellular  animals  the 
nutritive  process  consists  in  the  ingestion  of  minute  particles  of 
organic  matter  by  the  cell.  The  rhizopods,  which  may  be  found  and 
studied  in  water  from  stagnant  pools  or  infusions,  when  observed 
under  the  microscope,  may  be  seen  to  send  out  short  protoplasmic 
processes,  the  pseudopodia,  by  means  of  which  they  gradually  flow 
about  any  foreign  particle  with  which  they  come  in  contact.  If  the 
ingested  particle  is  of  an  inorganic  nature  and  indigestible,  it  will  be 
again  extruded  after  a  varying  period.  If,  however,  the  ingested  sub- 
stance is  of  a  nature  which  can  be  utilized  in  the  nutrition  of  the  proto- 
zoon,  it  is  rapidly  surrounded  by  a  small  vacuole  within  which  it  is 
gradually  dissolved  and  becomes  a  part  of  the  cellular  protoplasm. 
This  digestion  within  the  unicellular  organism  is  probably  due  to  a 
proteolytic  enzyme  2  which  acts  in  the  presence  of  a  weakly  alkaline 

^Metchnikoff,  "L'lmmunite  dans  les  maladies  infectueuses. " 
2  Mouton,  Ann.  de  Finst.  Pasteur,  xvi,  1902. 

330 


PHAGOCYTOSIS  331 

reaction.     This  has  been  shown  by  the  actual  extraction,  from  amebae, 
of  a  trypsin-like  ferment. 

As  we  proceed  higher  in  the  scale  'of  the  animal  kingdom,  we  find 
that  this  power  of  intracellular  digestion,  while  not  uniformly  an 
attribute  of  all  the  body  cells,  is  still  well  developed  and  a  necessary 
physiological  function  of  certain  cells  which  have  retained  primitive 
characters.  In  animals  like  the  coelenterata,  in  which  there  are  two 
cell  layers,  an  entoderm  and  an  ectoderm,  the  ectodermal  cells  have  lost 
the  power  of  intracellular  digestion,  while  the  entodermal  cells  are 
•  still  able  to  ingest  and  digest  suitable  foreign  particles.  It  is  only  as 
we  proceed  to  animals  of  a  much  higher  organization  that  the  function 
of  cell  ingestion  of  crude  food  is  entirely  removed  from  the  process  of 
general  nutrition.  Nevertheless,-  in  these  animals  also,  the  actual  cell 
ingestion  of  foreign  particles  occurs,  but  it  is  now  limited  entirely  to 
a  definite  group  of  cells.  In  the  higher  animals  and  in  man,  this 
function  of  phagocytosis  is  limited  to  the  white  blood  cells  of  the 
circulation,  or  leucocytes,  to  certain  large  endothelial  cells  lining  the 
serous  cavities  and  blood-vessels,  and  to  cells  of  a  rather  obscure 
origin  which  contribute  to  the  formation  of  giant  cells  within  the 
tissues.  A  convenient  division  of  these  phagocytic  cells  is  that  into 
11  wandering  cells"  and  " fixed  cells."  The  wandering  cells  are  the 
polymorphonuclear  leucocytes,  called  "microphages"  by  Metchnikoff, 
and  certain  large  mononuclear  elements  or  ' '  macrophages. "  Fixed 
cells,  also  called  macrophages  by  Metchnikoff  and  possessing  the  power 
of  ameboid  motion,  include  the  cells  lining  the  serous  cavities,  and  the 
blood  and  lymph  spaces.  The  small  lymphocytes,  so  far  as  we  know, 
have  no  phagocytic  functions. 

In  studying  the  cellular  activities  which  come  into  play  whenever 
foreign  material  of  any  description  gains  entrance  into  the  animal 
body,  a  definite  reaction  on  the  part  of  the  phagocytic  cells  may  be 
observed.  When  we  inject  into  the  peritoneal  cavity  of  a  guinea-pig 
a  small  quantity  of  nutrient  broth,  and  examine  the  exudate  within  the 
cavity  from  time  to  time,  we  can  observe  at  first  a  diminution  from  the 
normal  of  the  cells  present  in  the  peritoneal  fluid.  This  may  be  due 
either  to  an  injury  of  the  leucocytes  by  the  injected  substance,  or  to 
an  actual  repellent  influence  which  the  injected  foreign  material  exerts 
upon  the  wandering  cells.3  Very  soon  after  this,  however,  the  exudate 
becomes  extremely  rich  in  leucocytes,  chiefly  of  the  polymorphonuclear 


3  Pierrallini,  Ann.  de  1'inst.  Pasteur,  1897. 


332  INFECTION  AND  IMMUNITY 

variety,  the  maximum  of  the  reaction  being  reached  about  eighteen  to 
twenty-four  hours  after  the  injection.  After  this,  there  is  a  gradual 
diminution  in  the  leucocytic  elements  until  the  fluid  in  the  peritoneal 
cavity  again  reaches  its  normal  condition.  It  is  plain,  therefore,  that 
the  presence  of  the  foreign  material  in  the  peritoneal  cavity  has,  after 
a  primary  repellent  action  upon  the  phagocytes,  attracted  them  in 
large  numbers  to  the  site  of  the  foreign  substance.  Such  repelling  or 
attracting  influences  upon  the  leucocytes  are  spoken  of  as  negative  or 
positive  chemotaxis.  The  reasons  for  chemotaxis  are  not  well  under- 
stood. In  the  case  of  bacteria,  which  chiefly  interest  us  in  the  present 
connection,  chemotactic  attraction  or  repulsion  is  intimately  dependent 
upon  the  nature  of  the  microorganism,  and  very  probably  has  a 
definite  relationship  to  its  virulence.  Whether  or  not  the  principles 
of  chemotaxis  may  serve  to  explain  the  hypo-  and  hyper-leucocytoses, 
observed  and  diagnostically  utilized  in  clinical  medicine,  is  by  no 
means  positive.  It  is  likely,  however,  that  the  two  phenomena  are 
closely  associated.  Levaditi  4  believed  that  he  obtained  some  evidence 
that  negative  chemotaxis  may  take  place  within  the  blood-vessels  when 
he  noticed  that  the  intravenous  injection  of  cholera  spirilla  into 
immunized  guinea-pigs  resulted  in  an  immediate  disappearance  of 
leucocytes  from  the  circulating  blood,  and  their  accumulation  in  the 
internal  organs.  On  the  other  hand,  this  may  possibly  be  more 
logically  explained  by  a  concentration  of  both  bacteria  and  leucocytes 
in  the  capillary  system  of  such  an  organ  as  the  liver,  as  it  is  known 
that  injected  bacteria  rapidly  disappear  from  the  general  circulation, 
but  may  be  demonstrated  in  the  various  organs  for  some  time  after 
injection. 

We  have  seen,  therefore,  that  the  invasion  of  the  animal  body  by 
foreign  material,  living  or  dead,  is  followed  by  a  prompt  response  on 
the  part  of  the  phagocytic  cells.  In  the  case  of  bacteria,  when  these 
are  deposited  in  the  subcutaneous  areolar  tissues,  the  inflammatory 
reaction  which  follows  brings  with  it  an  emigration  of  microphages 
(polynuclear  leucocytes)  from  the  blood-vessels — and  these  are  the 
so-called  pus  cells.  When  the  injection  of  bacteria  is  intraperitoneal, 
after  a  primary  diminution,  there  is  an  increase  of  leucocytes  in  the 
peritoneal  cavity  which  soon  results  in  the  formation  of  a  copious 
turbid  exudate.  If  the  pus  of  an  abscess  or  the  exudate  from  an 
infected  peritoneum  is  examined  microscopically,  it  will  be  seen  that 
many  of  the  microphages  have  taken  bacteria  into  their  cytoplasm. 

4  Levaditi,  Presse  med.,  1900. 


PHAGOCYTOSIS  333 

That  fully  virulent  living  bacteria  can  be  so  taken  up  has  been  vari- 
ously proven.  The  phagocytosis  is,  therefore,  not  simply  a  removal  of 
the  dead  bodies  of  bacteria  previously  killed  by  the  body-fluids,  but 
represents  an  actual  attack  upon  living  and  fully  virulent  micro- 
organisms. That  the  ingested  bacteria  are  often  alive  after  ingestion 
is  proved  by  the  fact  that  the  injection  of  exudate  containing,  so  far 
as  can  be  determined,  only  intracellular  bacteria,  has,  in  several 
instances,  been  found  to  give  rise  to  infection. 

After  the  bacteria  have  remained  for  some  time  within  the  cyto- 
piasm  of  the  leucocyte,  vacuoles  may  be  seen  to  form  about  them, 
similar  to  those  mentioned  in  discussing  the  digestive  processes  of 
amebae.  If  the  preparations  are,  at  this  stage  or  later,  stained  with  a 
one-per-cent  solution  of  neutral  red,  it  will  be  found  that  the  bacteria, 
colorless  under  normal  conditions,  will  be  stained  pink,  an  evidence  of 
their  beginning  disintegration.  At  a  later  stage  in  the  process  of  intra- 
cellular digestion,  the  bacteria  will  lose  their  form,  and  appear  swollen, 
granular,  and  vacuolated,  and  finally  will  be  110  longer  distinguishable. 
If,  on  the  other  hand,  the  ingestion  of  bacteria  brings  about  the  death 
of  a  leucocyte,  the  neutral  red  will  not  stain  the  bacteria,  the  digestive 
vacuoles  will  not  form,  and  the  leucocyte  itself  will  disintegrate. 

It  must  not  be  forgotten,  however,  that  not  all  microorganisms  are 
equally  susceptible  to  phagocytosis.  Some  may  resist  ingestion  more 
energetically  than  others  by  agencies  not  fully  understood.  Others 
again,  like  the  tubercle  bacillus  and  the  anthrax  bacillus  for  instance, 
may,  after  ingestion,  oppose  great  difficulties  to  intracellular  digestion. 

To  a  certain  extent,  moreover,  the  variety  of  the  bacterium  deter- 
mines the  variety  of  phagocyte  attracted  to  the  point  of  invasion.  In 
the  cases  of  most  of  the  bacteria  of  acute  diseases,  the  microphages  or 
polymorphonuclear  leucocytes  are  the  ones  upon  which  the  brunt  of 
the  battle  devolves.  Other  invaders,  like  the  Bacillus  tuberculosis, 
blastomyces,  and  others,  find  themselves  opposed  chiefly  by  the  macro- 
phages.  Cells  of  animal  origin,  such  as  the  dead  or  injured  cells  of  the 
animal's  own  body  or  the  cells  of  other  animals  artificially  introduced, 
are  ingested  by  macrophages.  This  is  true  also  of  many  parasites  of 
animal  nature. 

It  is  clear,  thus,  that  the  process  of  phagocytosis  is  a  universal 
response  on  the  part  of  the  body  to  the  invasion  of  foreign  particles  of 
dead  material,  of  alien  cells,  and  of  living  microorganisms.  It  remains 
to  be  shown  upon  what  basis  this  process  may  be  regarded  as  an 
essential  feature  in  protecting  the  body  against  infection. 


334  INFECTION   AND   IMMUNITY 

The  numerous  researches  of  Metchnikoff  have  brought  out  the 
important  fact  that  phagocytosis  is  regularly  more  active  in  cases  in 
which  the  infected  animal  or  human  being  eventually  recovers.  In 
animals,  furthermore,  which  show  a  high  natural  resistance  against 
any  given  microorganism,  phagocytosis  is  decidedly  more  energetic 
than  it  is  in  animals  more  susceptible  to  the  same  incitant.  Thus, 
experimenting  with  anthrax  infection  in  rats,  Metchnikoff  was  able  to 
show  that,  in  these  animals,  a  decidedly  more  rapid  and  extensive 
phagocytosis  of  anthrax  bacilli  takes  place  than  in  rabbits  and  guinea- 
pigs  and  other  animals  which  are  delicately  susceptible  to  this  infec- 
tion. While  different  interpretations  have  been  attached  to  this 
phenomenon,  its  actual  occurrence  may  be  accepted  as  a  proven  fact. 

In  his  later  investigations,  furthermore,  Metchnikoff  was  able  to 
show  that  a  direct  parallelism  existed  between  the  development  of 
immunity  in  an  artificially  immunized  animal  and  the  phagocytic 
powers  of  its  white  cells.  He  showed  that  rabbits  artificially  im- 
munized to  anthrax,  responded  to  anthrax  infection  by  a  far  more 
active  phagocytosis  than  did  normal,  fully  susceptible  animals  of  the 
same  species. 

It  is  quite  impossible,  in  the  space  allotted,  to  recount  the  many 
similar  experiments  by  which  the  accuracy  of  these  observations  has 
been  confirmed.  While  few  bacteriologists  at  the  present  day  harbor 
any  doubt  as  to  the  truth  of  these  contentions,  the  fundamental  dif- 
ferences between  the  conclusions  drawn  from  these  various  phenomena 
by  the  school  of  Metchnikoff  and  by  that  of  the  German  workers  may 
be  clearly  stated  as  follows :  Metchnikoff  believes  that  phagocytosis  is 
the  cardinal  factor  which  determines  immunity,  while  Pfeiffer  and 
others  maintain  that  the  determining  factors  upon  which  recovery  or 
lethal  outcome  depends,  lie  in  the  fluids  of  the  body,  the  serous  exudate 
and  its  contents  of  immune  body  and  complement,  while  the  phago- 
cytosis occurring  coincidently,  is  merely  a  means  of  removal  of  the 
bacteria  after  the  outcome  has  already  been  decided. 

In  the  further  developments  of  his  theory,  Metchnikoff  has  claimed 
that  the  immune  body  and  complement — the  presence  of  which  in 
blood  serum  and  exudates  he  by  no  means  overlooks — are  derivatives 
of  the  leucocytes. 

The  immune  body  or  "fixator,"  as  Metchnikoff  has  named  it,  has 
been  shown  by  Wassermann  and  Takaki 5  to  be  most  plentiful  in  the 

5  Wassermann  und  Takaki,  Berl.  klin.  Woch.,  1898. 


PHAGOCYTOSIS  335 

spleen,  lymph  nodes,  and  bone  marrow  of  animals — all  of  them  organs 
in  which  large  collections  of  leucocytic  elements  are  found.  Metch- 
nikoff's  opinions  as  to  the  leucocytic  origin  of  the  complement,  or 
"cytase,"  have  found  support  in  the  experiments  of  Levaditi,6  who 
was  able  to  demonstrate  the  absence  of  complement  in  blood  plasma, — 
i.e.,  where  no  destruction  of  leucocytes  had  taken  place — and  in  those 
of  Cantacuzene,7  who  showed  that  cholera-immune  guinea-pigs  would 
succumb  to  intraperitoneal  injection  of  these  bacteria  when  the 
diapedesis  of  leucocytes  had  been  prevented  by  the  administration  of 
opium. 

The  chapter  of  phagocytosis  in  its  relation  to  bacterial  immunity  is 
by  no  means  closed.  The  problems  involved  in  it  are  intricate  and 
will  require  much  further  study.  The  subsequent  sections  upon 
opsonins,  aggressins,  and  upon  leucocyte  extract,  incorporate  the  more 
recent  studies  which  may  be  said  to  have  followed  logically  in  the 
footsteps  of  Metchnikoff's  work. 

6  Levaditi,  Presse  med.,  1900. 

T  Cantacuzcne,  Ann.  de  1'inst.  Pasteur,  1897. 


CHAPTER  XVIII 

OPSON1NS  AND  VACCINE  THERAPY.     LEUCOCYTIC  SUBSTANCES. 
NON-SPECIFIC  PROTEIN  THERAPY.     VIRULENCE 

OPSONINS 

ALTHOUGH  the  theories  of  immunity  are,  as  we  have  stated,  gen- 
erally classified  as  the  humoral  and  the  cellular  or  phagocytic  theories, 
the  separation  has  never,  even  in  the  minds  of  the  warmest  partisans, 
been  an  absolute  one.  Thus,  Buchner  and  his  successors  looked  for 
the  origin,  first,  of  alexin,  then  of  antibody,  in  the  leucocytes,  and 
Metchnikoff  attributed  to  immune  serum  the  quality  of  stimulating 
the  leucocytes  (stimulins)  to  increased  phagocytosis.  The  serum, 
according  to  Metchnikoff,  acted,  not  directly  upon  the  bacteria,  in  the 
nature  of  bactericidal  or  lytic  substances,  but  rather  upon  the  leuco- 
cytes, preparing  or  arming  these  for  the  fray.  Denys  and  Leclef1 
were  the  first  definitely  to  oppose  this  view.  These  authors,  'on  the 
basis  of  experiments  done  upon  streptococcus  immunity  in  rabbits, 
came  to  the  conclusion  that  the  serum  aided  phagocytosis  rather  by  its 
action  upon  the  bacteria  than  by  its  influence  upon  the  leucocytes. 

Wright2  in  1903  and  1904  undertook  a  systematic  study  of  the 
relation  of  the  blood  serum  to  phagocytosis,  in  a  series  of  careful 
experiments.  Using  his  own  modifications  of  the  technique  of  Leish- 
man,3  he  first  determined  the  direct  dependence  of  phagocytosis  upon 
some  substance  contained  in  the  blood  serum.  He  further  proved 
conclusively  that  this  serum  component  acts  upon  the  bacteria  directly 
and  not  upon  the  leucocytes,  is  bound  by  the  bacteria,  and  renders  them 
subject  to  phagocytosis.  The  presence  of  these  substances  in  sera, 
furthermore,  which  appear  entirely  free  from  bactericidal  or  lytic 
bodies,  and  the  thermolabile  character  of  the  substances  (60°  for  ten 
or  fifteen  minutes  destroys  them)  seemed  to  exclude  their  identity  with 
the  immune  bodies  of  other  authors. 


1  Denys  et  Leclef,  La  cellule,  xi,  1895. 

2  Wright  and  Douglas,  Proe.  Royal  Soc.  London,  Ixxii,  1904. 
'Leishman,  Brit.  Med.  Jour.,  i,  1902. 

336 


OPSONINS  AND  VACCINE  THERAPY  337 

Because  of  their  action  in  preparing  the  bacteria  for  ingestion  by  the 
leucocytes,  he  named  those  bodies  "opsonins"  (oif/uvtiv,  to  prepare 
food). 

Neufeld  and  Rimpau4  soon  after,  and  independently  of  Wright, 
described  similar  substances  in  the  blood  serum  of  streptococcus  and 
pneumococcus  immune  animals,  which  they  called  bacteriotropins. 
Because  of  their  greater  thermostability  it  is  not  yet  possible  to 
identify  these  bacteriotropins  absolutely  with  the  opsonins. 

The  importance  of  these  opsonic  substances  in  immunity  was 
shown  by  Wright5  in  a  series  of  experiments  in  which  he  determined 
that  in  persons  ill  with  staphylococcus  or  tubercle-bacillus  infections, 
the  phagocytic  powers  were  relatively  diminished  toward  these 
microorganisms,  but  could  be  specifically  increased  upon  active  im- 
munization with  dead  bacteria  or  bacterial  products. 

The  results  of  Wright  have  been  confirmed  and  elaborated  by 
numerous  workers. 

The  diminished  power  of  leucocytes  to  take  up  bacteria  without 
the  cooperation  of  serum  was  demonstrated,  after  Wright,  by  Hek- 
toen  and  Ruediger,0  who  worked  with  gradually  increasing  dilutions 
of  serum.  The  contention  of  the  Wright  school,  however,  that 
leucocytes  are  entirely  impotent  for  phagocytosis  without  the  aid 
of  serum,  can  not  be  regarded  as  proven,  in  face  of  the  work  of 
Lohlein7  and  others  who  have  observed  phagocytosis  on  the  part 
of  washed  leucocytes. 

The  specificity  of  opsonins  and  their  multiplicity  in  a  given 
serum  were  shown  mainly  by  the  work  of  Bullock  and  Atkin,8 
Hektoen  and  Ruediger,9  and  Bullock  and  Western.10  These  authors 
showed  that  the  opsonic  substances  in  sera  could  be  absorbed  out 
of  the  sera,  one  by  one,  by  treatment  with  various  species  of  bacteria, 
a  procedure  analogous  to  the  method  of  absorption  used  in  the 
study  of  agglutinins. 

The  increase  of  phagocytic  power  demonstrated  by  Wright  in 
immune  sera  naturally  led  to  the  question  whether  this  depended 

^Neufeld  und  Rimpau,  Deut.  med.  Woch.,  xl,  1904. 

c  Wright  and  Douglas,  Proc.  Eoy.  Soc.,  London,  Ixxiv,  1905. 

*Hcktoen  and  Euediger,  Jour.  Inf.  Dis.,  ii,  1905. 

7  Lohlein,  Ann.  de  Pinst.  Pasteur,  1905  and  1906. 

8  Bullock  and  AtJcin,  Proc.  Eoy.  Soc.,  London,  Ixxiv,  1905. 

9  Hektoen  and  Ruediger,  loc.  cit. 

10  Bullock  and  Western,  Proc.  Roy.  Soc.,  loc.  cit. 


338  INFECTION  AND  IMMUNITY 

merely  upon  an  increase  of  the  normal  opsonins  or  whether  the 
newly  formed  immune  opsonins  were  entirely  different  substances. 
The  greater  thermostability  of  the  opsonins  in  immune  sera  seemed, 
at  first,  to  support  the  latter  view.  Dean,11  however,  showed  that 
not  all  of  the  normal  opsonins  are  thermolabile  and  that,  by  absorp- 
tion experiments,  bacteria  treated  with  normal  sera  could  be  pre- 
vented from  taking  up  opsonins  from  immune  sera.  These  facts 
seem  to  point  strongly  toward  the  identity  of  normal  and  immune 
opsonic  substances. 

Further  study  of  the  opsonins  has  led  to  numerous  other  ques- 
tions regarding  their  structure,  their  relation  to  other  immune 
bodies,  etc.,  which  are  largely  still  in  the  stage  of  controversy,  and 
for  which  the  original  monographs  must  be  consulted. 

The  controversial  questions  may  be  briefly  reviewed  as  follows: 

As  stated  above,  Wright  believed  originally  that  the  bodies  dis- 
covered by  him  in  normal  sera,  the  "normal  opsonins,"  in  other 
words,  were  distinct  bodies  that  could  not  be  identified  with  either 
the  complement  or  antibodies  present  in  serum.  Neuf  eld  and  Hiine,12 
Levaditi  and  Inmann,13  and  others,  on  the  other  hand,  maintain 
that  the  opsonic  action  of  normal  serum,  at  least,  is  intimately 
related  to  the  complement  contents  of  such  serum. 

They  base  this  contention  not  only  upon  the  thermolability  of 
normal  opsonins,  but  also  upon  the  fact  that  opsonin  may  be  removed 
from  normal  serum  at  the  same  time  as  complement  by  the  method 
of  complement  fixation,  detailed  in  another  section  (see  pp.  295 
and  315 ).14 

The  contention  of  Wright  that  the  thermostable  opsonic  sub- 
stances of  immune  serum  are  distinct  bodies,  not  identical  with  the 
amboceptors,  is  supported  by  the  work  of  Hektoen,15  Neufeld  and 
Topfer,16  and  others.  The  problem,  however,  can  by  no  means  be 
regarded  as  finally  settled,  since  other  workers,  notably  Levaditi, 
are  inclined  to  identify  the  immune  opsonins  with  lytic  amboceptors. 

As  to  the  structure  of  the  opsonic  substances,  moreover,  differ- 


11  Dean,  Proc.  Eoy.  Soc.,  London,  Ixxvi,  1905. 

12  Neufeld  and  Hune,  Arb.  a.  d.  kais.  Gesundheitsamt,  xxv. 

13  Levaditi  and  Inmann,  Compt.  rend,  de  la  soc.  de  biol.,  62,  1907. 

14  Levaditi,  Presse  medicale,  70,  1907. 
15Helctoen,  Jour,  of  Inf.  Dis.,  iii,  1906. 

16  Neuf eld  und  Topfer,  Cent.  f.  Bakt.,  xxxviii,  1905. 


OPSONINS  AND  VACCINE  THERAPY 


339 


ences  of  opinion  still  exist.  Hektoen  and  Ruediger17  who  have 
investigated  the  question  attribute  to  opsonins  a  complex  constitu- 
tion. They  believe  them  to  possess  a  thermostable  haptophore  group 
and  a  thermolabile  "opsonophore"  group  and  that  heating  beyond 
a  definite  temperature  converts  the  opsonins  into  opsonoids  by 
destruction  or  alteration  of  the  ' '  opsonophore "  group.  This  view 
is  not  shared  by  all  workers  and  has  been  disputed  by  Bullock 
and  Atkin.18 

The  Technique  of  Wright.— The  three  factors  necessary  for  the 
performance  of  an  opsonic  test  are  (1)  the  blood  serum  to  be  tested; 
(2)  an  even  emulsion  of  bacteria,  and  (3)  leucocytes. 


FIG.  40. — WRIGHT'S  CAPSULE  FOR  COLLECTING  BLOOD. 

(1)  Blood  serum  is  obtained  by  bleeding  from  the  finger  and 
receiving  the  blood  into  glass  capsules  (Fig.  40).    These  are  sealed 
at  both  ends;  the  blood  is  allowed  to  clot;  and  the  separation  of 
serum  is  hastened  by  a  few  revolutions  of  a  centrifuge. 

(2)  The  bacterial  emulsion  is  obtained  by  rubbing  up  a  few 
loopfuls   of   a   twenty-four-hour   slant    agar   culture   with   a   little 
physiological  salt  solution  (0.85  per  cent)  in  a  watch  glass.    A  very 


FIG.  41. — PIPETTE  FOR  OPSONIC  WORK. 

small  amount  of  salt  solution  is  used  at  first  and  more  is  gradually 
added,  drop  by  drop,  as  the  emulsion  becomes  more  even.  The  final 
breaking  up  of  the  smaller  »clumps  is  best  accomplished  by  cutting 
off  very  squarely  the  end  of  a  capillary  pipette,  placing  it  perpen- 
dicularly against  the  bottom  of  the  watch  glass,  and  sucking  the 
emulsion  in  and  out  through  the  narrow  chink  thus  formed. 
(Fig.  41.) 


17  Hektoen  and  Euediger,  Jour,  of  Inf.  Dis.,  ii,  1905. 

18  Bullock  and  Atldn,  Proc.  Koyal  Soc.,  Ixxiv,  1905. 


340  INFECTION  AND  IMMUNITY 

Emulsions  of  tubercle  bacilli  are  more  difficult  to  make.  The 
bacilli  filtered  off  in  the  manufacture  of  old  tuberculin  are  com- 
monly used.  These  are  washed  in  salt  solution  on  the  filter,  and 
are  then  scraped  off  and  sterilized.  They  are  then,  in  a  moist 
condition,  placed  in  a  mortar  and  thoroughly  ground  into  a  paste. 
While  grinding,  salt  solution  1.5  cent)  is  gradually  added  until  a 
thick  emulsion  appears.  This  emulsion  may  be  diluted  and  larger 
clumps  separated  by  centrifugalization. 

(3)  The  leucocytes  are  obtained  by  bleeding  from  the  ear  or 
finger  directly  into  a  solution  containing  eighty-five  hundredths  per 
cent  to  one  per  cent  of  sodium  chlorid  and  five-tenths  to  one  and 
five-tenths  per  cent  of  sodium  citrate.  Ten  or  fifteen  drops  of  blood 
to  5  or  6  c.c.  of  the  solution  will  furnish  sufficient  leucocytes  for  a 
dozen  tests.  This  mixture  is  then  centrifugalized  at  moderate  speed 
for  five  to  six  minutes.  At  the  end  of  this  time,  the  corpuscles  at 
the  bottom  of  the  tube  will  be  covered  by  a  thin  grayish  pellicle, 


FIG.  42. — PIPETTE  WITH  THREE  SUBSTANCES,  CORPUSCLES,  BACTERIA  AND  SERUM, 

AS  FIRST  TAKEN  UP. 

the  buffy  coat,  consisting  chiefly  of  leucocytes.  These  are  pipetted 
off  with  a  capillary  pipette  (by  careful  superficial  scratching  move- 
ments over  the  surface  of  the  buffy  coat). 

There  being,  of  course,  no  absolute  scale  for  phagocytosis,  when- 
ever an  opsonin  determination  is  made  upon  an  unknown  serum, 
a  parallel  control  test  must  be  made  upon  a  normal  serum.  This 
normal  is  best  obtained  by  a  "pool"  or  mixture  of  the  sera  of  five 
or  six  supposedly  normal  individuals. 

The  three  ingredients — serum,  bacterial  emulsion,  and  leucocytes 
—having  thus  been  prepared,  the  actual  test  is  carried  out  as  follows : 
Capillary  pipettes  of  about  six  or  seven  inches  in  length  and  of 
nearly  even  diameter  throughout,  are  made.  These  are  fitted  with 
a  nipple  and  a  mark  is  made  upon  them  with  a  grease-pencil  about 
2  to  3  cm.  from  the  end  (Fig.  42).  Corpuscles,  bacteria,  and  serum 
arc  then  successively,  in  the  order  named,  sucked  into  the  pipette 
up  to  the  mark,  being  separated  from  each  other  by  small  air- 
bubbles.  Equal  quantities  of  each  having  thus  been  secured,  they 
are  mixed  thoroughly  by  repeatedly  drawing  them  in  and  out  of 
the  pipette  upon  a  slide.  The  mixture  is  then  drawn  into  the  pipette ; 


OPSONINS  AND  VACCINE  THERAPY  341 

the  end  is  sealed;  and  incubation  at  37.5°  is  carried  on  for  an 
arbitrary  time,  usually  fifteen  to  thirty  minutes.19  The  control  with 
normal  serum  is  treated  in  exactly  the  same  way.  After  incubation 
the  end  of  the  pipette  is  broken  off,  the  contents  are  again  mixed, 
and  smears  are  made  upon  glass  slides  in  the  ordinary  manner  of 
blood  smearing.  Staining  may  be  done  by  .Wright's  modification 
of  Irishman's  stain,  by  Jcnner's,  or  by  any  other  of  the  usual  blood 
stains.  In  these  smears,  then,  the  number  of  bacteria  contained  in 
each  leucocyte  is  counted.  The  contents  of  about  eighty  to  one 
hundred  cells  are  usually  counted  and  an  average  is  taken.  This 
average  number  of  bacteria  in  such  leucocytes  is  spoken  of  as  the 
"phagocytic  index."  The  phagocytic  index  of  the  tested  serum, 
divided  by  that  of  the  " normal  pool"  (control)  serum,  gives  the 
"opsonic  index." 

Another  method  of  estimating  the  opsonic  content  of  a  given 
blood  serum  has  been  contributed  by  Simon,  Lamar,  and  Bispham.20 
These  authors  employed  dilutions  both  of  the  patient's  serum  and 
of  normal  serum  ranging  from  one  in  ten  to  one  in  one  hundred. 
With  these  dilutions,  they  carry  out  opsonic  experiments  with  bac- 
terial emulsions  and  washed  leucocytes  in  the  same  way  as  this  is 
done  in  the  Wright  method,  except  that  they  recommend  the  employ- 
ment of  thinner  bacterial  emulsions  than  are  usually  employed  in 
the  former  method.  In  examining  their  slides,  they  do  not  estimate 
the  number  of  bacteria  found  within  the  leucocytes,  but  rather  the 
percentage  of  leucocytes  which  actually  take  part  in  the  phagocytic 
process,21  i.e.,  which  contain  bacteria. 

By  the  same  method  of  dilution,  they  determine  what  they  have 
called  "the  opsonic  coefficient  of  extinction,"  a  phrase  which  is  used 
to  express  the  degree  of  dilution  of  the  serum  at  which  no  further 
phagocytosis  takes  place.  They  claim  for  their  methods  the  more 
delicate  determination  of  variations  in  opsonic  power.  The  method 
has  not  been  sufficiently  used  to  permit  the  expression  of  an  opinion 
as  to  its  value. 

The  Vaccine  Therapy  of  Wright. — In  connection  with  his  more 
theoretical  work  upon  opsonins,  Wright  has  laid  much  stress  upon 


19  For  the  purpose  of  incubation,  specially  constructed  water  baths,  marketed 
under  the  name  of  "opsonizers, "  may  be  used. 

™  Simon,  Lamar,  and  Bispltam,  Jour.  Exp.  Med.,  viii,  1906. 
21  Simon  and  Lamar,  Johns  Hopkins  Hosp.  Bull.,  xvii,  1906. 


342  INFECTION   AND   IMMUNITY 

the  value  of  active  immunization  in  the  treatment  of  infectious  dis- 
eases. Beginning  his  work  with  staphylococcus  and  tubercle-bacillus 
infections,  he  has  extended  his  methods,  with  the  aid  of  many  col- 
laborators, to  gonococcus,  streptococcus,  pneumococcus,  and  a 
number  of  other  bacterial  infections.  In  all  these  cases,  when  pos- 
sible, he  uses  for  therapeutic  purposes  a  so-called  "autogenous 
vaccine"  which  is  made  with  the  bacteria  isolated  from  the  patient 
himself.  In  the  case  of  tubercle-bacillus  infections,  lie  uses  for 
treatment  the  new-tuberculin-bacillary-emulsion  of  Koch.  The  pro- 
duction of  vaccine  is,  according  to  Wright,  as  follows : 

Production  of  Vaccines. — After  isolation  of  the  organisms  from  the 
patient,  cultures  are  made  with  a  view  of  obtaining  considerable 
amounts  of  bacterial  growth.  In  making  vaccines  with  poorly  grow- 
ing organisms,  large  surfaces  must  be  inoculated.  Organisms  are 
best  grown  for  this  purpose  upon  the  surface  of  agar  or  glucose 
agar  (the  enrichment  of  the  agar  with  sugar  or  acetic  fluid,  etc., 
depending  upon  the  cultural  requirements  of  the  organism  in  ques- 
tion), in  square  eight-ounce  medicine  bottles  laid  upon  their  sides. 
This  furnishes  a  large  area  for  inoculation.  After  sufficient  growth 
has  taken  place  upon  the  agar,  two  or  three  cubic  centimeters  of 
sterile  normal  salt  solution  are  introduced  into  the  bottles  with  a 
sterile  pipette.  With  this  the  growth  is  gently  washed  off  the 
surface  of  the  agar,  more  salt  solution  gradually  being  added  as 
necessary.  The  emulsification  may  be  facilitated  by  gently  scraping 
the  growth  off  the  medium  by  means  of  a  flexible  platinum  loop. 
This  thick  bacterial  emulsion  is  then  pipetted  out  of  the  bottles, 
during  which  process  an  equalization  of  the  emulsion  can  be  attained 
by  repeated  sucking  in  and  out  with  the  pipette.  The  emulsion  is 
then  placed  in  a  sterile  test  tube  which  may  then  be  drawn  out 
at  its  open  end  into  a  capillary  opening.  It  is  a  point  of  practical 
importance  that,  in  preparing  such  capsules  out  of  a  test  tube,  a 
few  inches  of  air  space  should  be  left  above  the  surface  of  the 
emulsion,  so  that  expansion  during  heating  may  not  blow  out  the 
top  of  the  glass  tube.  A  dozen  or  so  of  sterile  glass  beads  may  be 
put  into  these  tubes  in  order  to  aid  in  emulsification.  Shaking  the 
beads  in  such  a  tube  will  help  in  breaking  up  small  clumps  of 
bacteria. 

The  emulsion  is  then  standardized;  that  is,  a  numerical  estima- 
tion of  bacteria  per  cubic  centimeter  must  be  made.  This  standard- 
ization is  best  done  before  sterilization,  because  during  the  latter 


OPSONINS  AND  VACCINE  THERAPY  343 

process  a  number  of  bacteria  may  be  broken  up,  and,  while  un- 
recognizable morphologically,  are,  nevertheless,  represented  in  the 
emulsion  by  their  products.  The  standardization  may  be  accom- 
plished by  highly  diluting  a  definite  volume  of  the  emulsion,  planting 
plates  with  definite  quantities  of  the  dilution,  and  counting  colonies. 
Wright  prefers,  as  more  exact,  an  enumeration  of  tfte  bacteria 
against  red  blood  cells.  This  is  done  in  the  following  way : 

A  little  of  the  emulsion  is  placed  in  a  watch  glass  and  from  it, 
with  a  pipette  as  used  in  the  estimation  of  the  opsonic  index,  one 
volume  is  taken  and  is  mixed  with  an  equal  volume  of  blood  from 
the  finger  and  two  or  three  volumes  of  salt  solution.  The  salt 
solution  is  added  in  order  to  dilute  the  red  cells  so  that  they  can 
be  conveniently  counted  and  to  prevent  clotting.  These  substances 
are  thoroughly  mixed  in  a  pipette  and  spread  upon  a  slide  as  in 
making  a  blood  smear,  and  as  even  and  uniform  a  smear  as  possible 
should  be  made.  They  are  then  stained  either  by  Jenner's  or 
Wright's  blood  stain. 

The  preparations  are  examined  with  an  oil-immersion  lens.  In 
order  to  limit  a  definite  microscopic  field,  it  is  convenient  to  use  an 
Ehrlich  diaphragm,  or  else,  in  lieu  of  this,  to  mark  a  circle  with 
a  blue  pencil  upon  the  lens  of  the  eye-piece.  The  red  blood  cells 
and  bacteria,  in  a  number  of  these  fields,  are  counted  and  the  ratio 
between  them  is  estimated.  Knowing  the  number  of  red  blood  cells 
to  the  cubic  millimeter  in  the  particular  blood  employed,  by  previous 
blood  count,  and  knowing  that  equal  volumes  of  blood  and  of  bac- 
terial emulsion  have  been  used  in  the  mixture,  it  is  easy  from  this 
ratio  to  ascertain  the  number  of  bacteria  contained  in  a  cubic  milli- 
meter of  the  original  emulsion.  Thus,  for  instance,  if  in  an  average 
of  twenty  fields  bacteria  are  to  red  blood  cells  as  two  is  to  one, 
and  the  blood  employed  contains  five  million  red  blood  cells  to  each 
cubic  millimeter,  then  a  cubic  millimeter  of  our  emulsion  contained 
ten  million  bacteria,  and  a  cubic  centimeter  one  thousand  times 
as  many. 

Special  centrifuge  tubes  with  graduated  narrow  tips  at  the  bot- 
tom have  been  suggested  by  Hopkins  for  vaccine  standardization. 
Bacteria  centrifugalized  up  to  a  certain  mark  represent  a  definite 
number  per  cu.  mm.  wnen  taken  up  in  a  given  volume  of  salt  solution. 

The  vaccine,  thus  standardized,  is  sterilized  at  60°  C.  for  one 
hour  for  several  days.  Its  sterility  is  then  controlled  by  culture 
and  animal  inoculation. 


344  INFECTION  AND   IMMUNITY 

From  this  stock  emulsion  small  quantities  may  be  drawn  off  and 
diluted  for  therapeutic  use. 

The  initial  dose  given  by  Wright  in  staphylococcus  infections, 
in  which  the  method  has  been  most  frequently  employed,  varies 
from  fifty  to  one  hundred  millions  of  bacteria.  In  working  with 
the  tubercle  bacillus,  the  ordinary  tuberculin  dosage  is  adhered  to. 

Wright,  in  his  work,  makes  use  of  the  opsonic  index  in  order  to 
estimate  changes  in  the  resistance  of  the  patient  against  the  given 
infection.  In  other  words,  he  bases  his  judgment  as  to  whether  the 
patient  is  improving  or  not,  upon  the  opsonic  power  of  the  patient's 
serum.  In  following  the  opsonic  index  of  a  patient  during  systematic 
treatment  with  vaccine,  Wright  has  found  definite  changes  upon 
the  basis  of  which  he  constructs  a  curve  of  opsonic  power.  Imme- 
diately after  the  injection  of  vaccine,  he  finds  that  there  is  a  brief 
period  during  which  the  opsonic  power  of  the  patient  is  depressed 
below  its  original  state.  This  he  calls  the  negative  phase.  The 
length  of  time  occupied  by  this  negative  phase  depends  both  upon 
the  condition  of  the  patient  and  upon  the  size  of  the  dose  given. 
It  is  usually  completed  within  twenty-four  hours.  After  this,  there 
is  a  gradual  rise  in  the  opsonic  power,  at  first  rapid,  later  more 
slow,  until  a  maximum  is  reached  after  a  varying  number  of  days. 
This  period  of  rise  represents  the  positive  phase.  The  second  inocula- 
tion with. vaccine  should,  according  to  Wright,  be  made  when  the 
opsonic  power  is  again  beginning  to  sink  after  the  highest  point  of 
the  positive  phase. 

The  facts  of  Wright's  investigations  have  been  given  in  the 
preceding  without  much  critical  consideration.  Those  features  which 
concern  themselves  with  the  proof  of  the  opsonic  properties  of 
normal  and  immune  serum  have  been  of  the  greatest  scientific 
importance  and  a  great  deal  of  benefit  has  accrued  from  the  renewed 
attention  turned  by  him  toward  methods  of  active  immunization  in 
human  beings.  Vaccine  therapy  in  many  conditions  has  come  to 
stay,  although  some  of  the  very  extravagant  earlier  claims  have  had 
to  fall  down.  It  is  also  pretty  certain  at  present  that  the  opsonic 
index  measurements  as  a  guide  to  treatment  are  of  very  little  value 
and  may  even  mislead.  Prophylactic  vaccination  in  various  diseases 
is  dealt  with  in  the  chapters  on  the  individual  infections. 

Leucocytic  Substances. — In  the  sections  upon  Phagocytosis  and 
Opsonins,  we  have  discussed  the  protective  action  exerted  by  the 
living  leucocytes  against  bacterial  infection  and  the  relation  of  these 


OPSONINS  AND  VACCINE  THERAPY  345 

cells  to  the  blood  serum;  furthermore,  that,  while  our  knowledge 
of  the  blood  serum,  as  developed  at  present,  shows  that  phagocytes 
may  be  aided  by  this  in  the  ingesti.on  of  bacteria,  the  subsequent 
digestion  of  the  germs,  and  possibly  the  neutralization  or  destruction 
of  their  intracellular  poisons,  is,  as  far  as  we  know,  largely  accom- 
plished by  the  unaided  phagocytic  cell.  It  is  an  obvious  thought, 
therefore,  that,  in  the  struggle  with  bacterial  invaders,  the  leucocytic 
defenders  might  be  considerably  re-enforced  if  they  were  furnished, 
as  directly  as  possible,  with  a  further  supply  of  the  very  weapons 
which  they  were  using  in  the  fight  with  Hiss22  conceived  the  plan 
of  injecting  into  infected  subjects  the  substances  composing  the  chief 
cells  or  all  the  cells  usually  found  in  exudates,  in  the  most  diffusible 
form  and  as  little  changed  by  manipulation  as  possible ;  and  he  also 
assumed  that  extracts  would  be  more  efficacious  than  living  leuco- 
cytes themselves,  since  if  diffusible  they  would  be  distributed  im- 
partially to  all  parts  of  the  body  by  the  circulatory  mechanism. 

The  method  of  obtaining  these  substances  as  used  both  in  animal 
experiments  and  in  the  treatment  of  human  subjects  is  at  present 
as  follows: 

Rabbits,  preferably  of  1,500  grams  weight  or  heavier,  receive 
intrapleural  injections  of  aleuronat.  This  is  prepared  by  making  a 
three  per  cent  solution  of  starch  in  meat-extract  broth,  without  heat- 
ing, and  adding  to  this,  after  the  starch  has  gone  into  thorough 
emulsion,  five  per  cent  of  powdered  aleuronat.  This  is  thoroughly 
mixed,  boiled  for  five  minutes,  and  filled  into  sterile  potato  tubes, 
20  c.c.  into  each  tube.  Final  sterilization  is  done  preferably  in  an 
autoclave.  The  rabbit  injections  are  carried  out  by  injecting  10  c.c. 
into  each  pleural  cavity  in  the  intercostal  spaces  at  the  level  of  the 
end  of  the  sternum,  in  the  anterior  axillary  line,  great  care  being 
exerted  to  avoid  puncturing  of  the  lungs.  The  rabbits  are  left 
for  twenty-four  hours,  at  the  end  of  which  time  a  copious  and  very 
cellular  exudate  will  have  accumulated  in  the  pleural  cavities.  This 
is  removed,  after  killing  the  animals  with  chloroform,  by  opening 
the  anterior  chest  wall  under  rigid  precautions  of  sterility,  and 
pipetting  the  exudate  into  sterile  centrifuge  tubes.  Immediate  cen- 
trifugalization  before  clotting  can  take  place  then  permits  the 
decanting  of  the  supernatant  exudate  fluid.  To  the  leucocytic  sedi- 
ment is  then  added  about  2  c.c.  of  sterile  distilled  water,  and  the 


-  Hiss,  Jour.  Med.  Ees.,  N.  S.,  xiv,  3,  1908. 


346  INFECTION   AND  IMMUNITY 

emulsion  is  thoroughly  beaten  up  with  a  stiff  bent  platinum  spatula. 
Smears  are  now  made  on  slides,  stained  by  Jenner's  blood  stain, 
and  examined  for  possible  bacterial  contamination.  It  is  well  also 
to  take  cultures.  Sterile  distilled  water  is  then  added  to  each  tube, 
about  twenty  volumes  to  one  volume  of  sediment,  and  the  tubes  are 
set  away  in  the  incubator  for  eight  hours.  At  the  end  of  this  time 
the  sterility  is  again  controlled  as  above,  and  further  extraction  in 
the  refrigerator  continued  until  the  extract  is  used. 

Experiments  by  Hiss  and  by  Hiss  and  Zinsser  showed  that  leu- 
cocytic  extracts  injected  into  animals  infected  with  various  or- 
ganisms exerted  a  distinct  though  not  very  powerful  therapeutic 
effect.  They  have  also  had  a  certain  degree  of  beneficial  effect  in 
human  beings  suffering  from  various  infections.  However,  it  is  our 
present  opinion,  based  chiefly  upon  the  researches  of  one  of  us  with 
Tsen,24  that  the  leucocytic  substances  act  in  more  or  less  the  same 
way  as  do  other  non-specific  proteins,  in  that  they  produce  an 
increased  leucocytosis  and  perhaps,  as  pointed  out  by  Jobling  and 
Petersen  in  another  connection,  may  lead  to  an  increased  discharge 
of  the  blood  of  various  proteolytic  and  other  enzymes. 

That  bactericidal  substances  can  be  extracted  from  leucocytes 
by  various  methods  has  been  repeatedly  shown  by  Schattenfroh, 
Pettersen,  Korschun,  and  others.25  The  researches  of  Pettersen  as 
well  as,  more  recently,  the  work  of  Zinsser,  have  shown  that  these 
"endolysins, "  as  Petterson  has  called  them,  have  a  structure  quite 
different  from  that  of  the  serum  bacteriolysins  in  that  they  are  not 
rendered  inactive  by  temperatures  under  80°  C.,  but,  when  once 
destroyed  by  higher  temperatures,  can  not  be  reactivated  either  by 
the  addition  of  fresh  serum  or  of  unheated  leucocyte  extracts.  The 
last-named  authors,  moreover,  have  shown  that  these  endocellular 
bactericidal  substances  are  not  increased  by  immunization,  the  quan- 
tity present  in  each  leucocyte  being  probably  at  all  times  simply 
sufficient  for  the  digestion  of  the  limited  number  of  bacteria  which 
can  be  taken  up  by  the  individual  leucocyte. 


23  Hiss,  Jour.  Med.  Kes.,  N.  S.,  xiv,  3,  1908. 

24  Zinsser  and  Tsen,  Jour,  of  Immun.,  2,  191 7. 

25  SchattciifroU,  Arch.  f.  Hyg.,  1897;  Pettcrxon,  Cent.  f.  P>aktv  I,  xxxix,  1905- 
and   ibid.,   xlvi,    1908;    Korschum,   Ann.   de   1'Inst.    Pasteur,    xxii,    1908;    Zinsser, 
Jour.  Med.  lies.,  xxii,  3,  1910. 


OPSONINS  AND  VACCINE  THERAPY  347 

NON-SPECIFIC  PROTEIN  THERAPY 

A  very  surprising-  development  of  the  last  ton  years  has  been 
the  observation  that  profound  physiological  reactions  accompanied 
by  occasional  therapeutic  benefit  in  infectious  diseases  has  followed 
the  intravenous  injection  of  bacterial  and  other  proteins  which  ap- 
parently had  no  specific  relationship  to  the  nature  of  the  infectious 
process.  The  first  observations  were  more  or  less  accidental,  in- 
cident to  attempts  by  various  writers  to  treat  diseases  like  typhoid 
fever  by  the  intravenous  injection  of  typhoid  bacilli.  Ichiwaka, 
Kraus,  Gay  and  others  injected  sensitized  and  unsensitized  typhoid 
bacilli  intravenously  into  patients  suffering  from  typhoid  fever,  ob- 
serving a  sudden  drop  of  temperature  with  chill,  and  frequent 
benficial  effects  on  the  course  of  the  disease.  It  was  soon  found 
that  similar  results  could  be  obtained  in  these  diseases  with  colon 
bacilli,  paratyphoid  bacilli,  etc.  Holler26  obtained  striking  results 
in  typhoid  fever  by  injecting  deutero-albumose.  Other  proteins  and 
proteose  substances  were  subsequently  used  by  many  observers,  and 
important  studies  on  the  theoretical  effects  of  the  injection  of  such 
substances  have  been  made  by  Jobling  and  Petersen.27  It  can  be 
regarded  as  quite  definite  that  these  reactions  are  entirely  non- 
specific. The  substances  used  have  been  typhoid  vaccine,  primary 
and  secondary  albumoses,  gonococcus  and  other  bacterial  vaccines, 
normal  serum,  leucocyte  extracts,  etc.,  etc.  The  method  has  been 
applied  to  a  great  many  definite  infections,  such  as  typhoid  fever, 
general  sepsis,  pneumonia,  gonorrheal  infections,  and  to  arthritis 
and  dermatological  lesions,  etc.  Miller28  has  used  typhoid  vaccines 
in  typhoid  fever  and  other  conditions,  and,  in  general,  concludes 
that  .there  can  be  little  doubt  that  in  a  limited  number  of  cases 
rapid  and  sometimes  permanent  beneficial  results,  are  obtained  after 
a  preliminary  slight  rise  of  temperature  and  subsequent  drop,  often 
with  a  chill.  He  also  concludes  that  if  an  amount  just  sufficient 
to  incite  a  chill  is  used,  that  is,  if  the  dosage  is  carefully  controlled, 
the  treatment  is  without  danger.  He  has  given  2,000  intravenous 
injections  of  typhoid  vaccine  in  the  Cook  County  Hospital,  without 
serious  consequences,  except  for  the  development  of  delirium  tremens 

~«  Holler,  Beit.  z.  Frank,  u.  Inkeft.,  6,  1917,  cited  from  Miller,  Jour.  A.  M.  A., 
76,  Jan.,  1921. 

"'Jobling  and  Petersen,  Jour.  Exper.  Med.,  20,  1914. 
**  Miller,  loc.  cit. 


348  INFECTION   AND   IMMUNITY 

in  some  alcoholics.  He,  however,  carefully  selected  his  cases.  As 
Petersen29  concludes,  non-specific  therapy  has  produced  definite  re- 
sults, though  the  eventual  determination  of  its  definite  value  cannot 
yet  be  made.  It  is  in  the  experimental  stage,  and  according  to 
Petersen29  concludes,  non-specific  therapy  has  produced  definite  re- 
sults, though  the  eventual  determination  of  its  definite  value  cannot 
yet  be  made.  It  is  in  the  experimental  stage,  and  according  to 
Petersen,  "its  usefulness  and  ultimate  range"  cannot  yet  be  fully 
judged. 

The  effects  of  the  injection,  as  analyzed  by  Petersen  from  his 
own  studies  and  a  study  of  the  literature,  are  as  follows:  After 
injection  of  the  more  powerful  and  active  substances,  there  is  at 
first  a  chill,  sweating,  and  a  definite  rise  of  temperature ;  there  is  a 
leucopasnia  followed  by  leucocytosis,  lowering  of  the  blood  pressure, 
and  changes  in  the  blood,  such  as  increase  in  fibrinogen,  a  rise  of 
enzyme  curve  and  an  increase  in  blood  sugar  and  antibodies.  The 
less  active  substances  produce  some  temperature,  a  slight  chill  and 
other  symptoms  mentioned,  to  a  lesser  degree.  The  beneficial  effects 
may  perhaps  be  to  some  extent  explained  by  the  increase  of  leu- 
cocytes and  of  enzymes,  and  Petersen  makes  a  point  of  the  fact 
that  if  the  method  is  to  exert  beneficial  effects,  it  is  probably  neces- 
sary to  use  it  early  in  the  disease.  We  are  not  in  any  position  at 
present  either  to  recommend  or  further  comment  upon  the  method, 
but  it  is  an  important  problem  for  laboratory  experimentation  and 
for  careful  clinical  application  in  the  hands  of  men  trained  in  ex- 
perimental studies. 

The  Problem  of  Virulence. — An  extremely  obscure  chapter  in  our 
knowledge  of  the  reaction  of  animals  and  man  against  infection  is 
the  one  dealing  with  the  questions  of  varying  pathogenicity  between 
different  bacterial  species  and  between  different  races  of  the  same 
microorganism.  We  know  that  certain  bacteria  may  be  injected  into 
an  animal  or  human  being  in  considerable  quantities,  without  pro- 
ducing anything  more  than  the  temporary  local  disturbance  follow- 
ing the  subcutaneous  administration  of  any  innocuous  material. 
Other  bacteria,  on  the  other  hand,  such  as  the  bacillus  of  anthrax 
or  the  bacillus  of  chicken  cholera,  injected  in  the  most  minute 
dosage,  may  give  rise  to  a  rapidly  fatal  septicemia.  Within  the 
same  species,  furthermore,  fluctuations  in  virulence  may  take  place 

29  Petersen,  Jour.  A.  M.  A.,  76,  January,  1921. 


OPSONINS  AND   VACCINE   THERAPY  349 

which  may  depend  upon  a  variety  of  influences  which  have  been 
discussed  in  another  section  and  need  not  be  recapitulated.  Suffice 
it  to  say  that  variations  in  the  susceptibility  of  inoculated  subjects 
do  not,  in  any  way,  furnish  a  sufficient  explanation  for  these 
phenomena. 

In  an  effort  to  cast  light  upon  this  subject,  Bail,  following  in  the 
footsteps  of  his  predecessors,  Kruse,30  Deutsch  and  Feistmantel,31 
has  formulated  his  so-called  ' i  aggressin-theory. " 

Bail32  was  first  led  to  the  formulation  of  his  theory  by  extensive 
researches  which  he  had  made  in  conjunction  with  Petterson33  into 
anthrax  immunity.  He  had  noted,  as  others  before  him  had,  that 
animals,  highly  susceptible  to  anthrax,  often  possessed  marked  bac- 
tericidal powers  against  this  bacillus.  When  such  animals,  whose 
serum  should  surely  be  capable  of  bringing  about  the  death  of,  at 
least,  a  few  hundred  anthrax  bacilli,  were  injected  with  doses  far 
less  than  this  number  they  nevertheless  succumbed  rapidly  and  the 
bacilli  multiplied  enormously  in  their  bodies.  He  argued  from  this 
that  the  injected  microorganisms  must  possess  some  weapon  whereby 
they  were  enabled  to  counteract  the  protective  forces  of  the  animal 
organism.  In  an  anthrax-immune  animal,  as  a  matter  of  fact,  no 
proliferation  of  bacteria  took  place  and  the  injected  germs  were 
rapidly  disposed  of  by  the  protective  forces,  foremost  of  which  was 
phagocytosis. 

The  theory  of  Bail34  contains  the  following  basic  principles:35 

Pathogenic  bacteria  differ  fundamentally  from  non-pathogenic 
bacteria  in  their  power  to  overcome  the  protective  mechanism  of 
the  animal  body,  and  to  proliferate  within  it.  They  accomplish  this 
by  virtue  of  definite  substances  given  off  by  them,  probably  in  the 
nature  of  a  secretion,  which  acts  primarily  by  protecting  them 
against  phagocytosis.  These  substances  (referred  to  by  Kruse  as 
"Lysins")  were  named  by  Bail,  *  *  Aggressins. "  The  production  of 
aggressins  by  pathogenic  germs  is  probably  absent  in  test-tube  cul- 
tures, or,  at  any  rate,  is  greatly  depressed  under  such  conditions, 

30  Kruse,  Ziegler  's  Beitrage,  xii,  1893. 

31  Deutsch  und  Feistmantel,  "Die  Impfstoffe  in  Sera,"  Leipzig,  1903. 

32  Bail,  Cent,  f .  Bakt.,  I,  xxvii,  1900,  and  xxxiii,  1902. 

83  Bail  und  Petterson,  Cent.  f.  Bakt.,  I,  xxxiv,  1903;  xxxv,  1904;  xxxvi,  1904. 
34  Bail,  Arch,  f .  Hyg.,  lii,  1905 ;  liii,  1905  ;  Wien.  klin.  Woch.,  xvii,  1905. 
85  Bail  und  Weil,  Wien.  klin.  Woch.,  ix,  1906;  Cent.  f.  Bakt.,  I,  xl,  1906;  xlii. 
1906. 


350  INFECTION   AND   IMMUNITY 

but  is  called  forth  in  the  animal  body  by  the  influences  encountered 
after  inoculation. 

These  aggressiiis  can  be  found,  according  to  Bail,  in  the  exudates 
about  the  site  of  inoculation  in  fatal  infections.  He  obtained  them, 
separate  from  the  bacteria,  by  the  centrifugation  and  subsequent 
decanting  of  edema  fluid,  and  pleural  and  peritoneal  exudates. 

Two  experimental  observations  are  brought  by  Bail  in  support 
of  the  truth  of  his  contentions.  In  the  first  place,  he  was  able  to 
show  that  fatal  infection  could  be  produced  in  animals  by  the 
injection  of  sublethal  doses  of  bacteria,  when  these  were  administered 
with  a  small  quantity  of  "aggressin."  He  inferred  from  this  that 
the  injected  aggressin  had  paralyzed  the  onslaught  of  phagocytic 
and  other  protective  agencies,  and  had  thus  made  it  possible  for 
the  bacteria  to  proliferate. 

The  second  experimental  support  of  Bail's  theory  consists  in  the 
successful  immunization  of  animals  with  aggressin.  Animals  were 
treated  with  aggressive  exudates,  from  which  all  bacteria  had  been 
removed  by  centrifugalization  and  which  had  been  rendered  sterile 
by  three  hours'  heating  to  60°  0.  and  addition  of  0.5  per  cent  phenol. 
Animals  so  treated  were  not  only  immune  themselves,  but  contained 
a  substance  in  their  serum  which  permitted  the  passive  immunization 
of  other  untreated  animals.  Bail  explained  this  by  assuming  the 
production  of  anti-aggressins  in  the  treated  subjects.  His  experi- 
ments and  those  of  his  pupils  were  conducted  with  the  typhoid  and 
dysentery  bacilli,  the  bacilli  of  chicken  cholera  and  of  plague,  the 
cholera  spirillum,  and  various  micrococci.  According  to  whether  a 
microorganism  is  capable  of  producing  an  aggressin  and  conse- 
quently of  invading  the  animal  body,  he  divides  bacteria  into  "pure 
parasites,"  "half  parasites,"  and  "saprophytes." 

The  theory,  of  Bail  has  been  attacked  chiefly,  by  Wassermann 
and  Citron,36  Wolff,37  and  Sauerbeck.38  The  criticism  which  these 
investigators  make  of  Bail's  views  has  succeeded  in  placing  the 
"aggressin"  theory  in  doubt.  It  is  claimed  by  them  that  much  of 
the  "aggressive"  character  of  Bail's  exudates  is  due  to  their  con- 
taining liberated  bacterial  poisons  (endotoxins).  This  they  have 
maintained  both  because  the  sterile  "aggressin"  exudates  could  be 
shown  to  possess  a  considerable  degree  of  toxicity  and  because  the 

M  Wassermann  and  Citron,  Deut.  med.  Woch.,  xxviii,  1905. 
«7  Wolff,  Cent.  f.  Bakt.,  I,  xxxviii,  1906, 
38  Sauerbeck,  Zeit.  f.  Hyg.,  Ivi,  1907, 


OP8ONINS  AND   VACCINE   THERAPY  351 

aggressive  action  could  be  duplicated  by  aqeous  extracts  of  bacteria. 
Citron,39  was  able  to  show,  by  the  Bordet-Gengou  method  of  com- 
plement fixation,  that  the  exudates  ,of  Bail  contained  quantities  of 
free  bacterial  receptors,  which,  in  taking  up  immune  body,  would 
neutralize  any  destructive  power  on  the  part  of  the  infected  animal. 

The  writer  in  conjunction  with  Dwyer40  has  done  certain  experi- 
ments which  seem  to  indicate  that  Bail's  aggressin  may  be  in  the 
nature  of  anaphylatoxin.  The  addition  of  such  anaphylatoxin  to 
bacteria  will  convert  a  sublethal  into  a  lethal  dose,  as  will  Bail's 
aggressin,  and  in  principle  the  manner  of  production  is  the  same. 
The  nature  of  the  immunity  produced  in  animals  by  Bail's  method 
of  treatment  is  less  easily  explained  and  less  exposed  to  adverse 
criticism.  Whatever  may  be  the  truth  about  the  possession  of  offen- 
sive weapons  on  the  part  of  bacteria,  it  is  certainly  a  fact  that 
microorganisms  differ  much  in  their  powers  of  defense  against  de- 
struction by  the  cells  in  sera  of  the  animal  body.  Virulent  bacteria 
are  not  destroyed  by  serum  or  agglutinated  or  taken  up  by  leucocytes 
as  easily  as  are  the  non-virulent.  In  some  cases  there  seems  to  be 
no  morphological  clue  to  the  reason  for  this.  In  other  cases,  like 
pneumococci,  Friedlander  bacilli  and  others,  there  is  a  bacterial 
capsule  which  seems  to  insulate  these  organisms  against  attack. 
Many  bacteria  lose  their  capsules  in"  the  non- virulent  stage  on 
culture  media,  but  form  them  within  the  animal  body  in  the  process 
of  infection.  Again,  bacteria  rendered  non-virulent  by  cultivation 
on  artificial  media  may  become  virulent,  inagglutinable,  and  more 
resistant  to  phagocytosis  when  cultivated  on  immune  sera  or  passed 
through  the  animal  body. 

Thus  the  power  to  invade  depends  possibly  upon  a  combination 
of  offensive  properties  and  defensive  qualities  on  the  part  of  the 
bacteria.  Added  to  this,  some  of  us  believe  that  the  reaction  between 
lytic  antibodies  and  the  bacterial  protein  may  produce  toxic  sub- 
stances which  poison  the  animal  body,  prevent  positive  chemotaxis, 
and  thereby  aid  the  invader. 

Again,  there  are  microorganisms  like  the  treponema  pallidum  in 
syphilis  where  adaptation  between  invader  and  host  seems  to  be  of 
such  a  nature  that  an  indifferent  reaction  against  the  invading 
organism  only  is  set  up. 

39  Citron,  Cent,  f .  Bakt.,  I,  xl,  1905 ;  xli,  1906 ;  and  Zeit.  f .  Hyg.  Hi,  1905. 

40  Zinsser  and  Dwyer,  Proceedings  of  the  Soc.  for  Exper.  Biol.  and  Med.,  1914, 
xi,  74-76. 


CHAPTER   XIX 

HYPERSUSCEPTIBILITY 

THE  phenomena  now  grouped  together  under  the  heading  of 
anaphylaxis  and  hypersusceptibility  have  but  recently  become  the 
subject  of  systematic  experimentation.  Nevertheless,  manifestations 
now  recognized  as  belonging  to  this  category  had  not  escaped  the 
attention  of  a  number  of  the  earlier  workers  in  immunity. 

Although  the  development  of  scientific  knowledge  of  hypersus- 
ceptibility has  concerned  itself  particularly  during  the  last  fifteen 
years  with  hypersusceptibility  phenomena  as  they  apply  to  antigenic 
substances,  we  must  deal  with  the  subject  a  little  more  compre- 
hensively than  this  at  the  present  day,  and  call  attention  to  the 
fact  that  clinicians  had  for  many  years  noticed  so-called  idiosyn- 
crasies against  drugs  of  various  kinds,  morphin,  strychnin,  arsenic, 
etc.,  etc.,  and  against  many  things  which  cannot  be  regarded  at  the 
present  time  as  antigens  in  the  sense  in  which  this  term  is  used  in 
immunological  literature. 

By  hypersusceptibility  in  general,  then,  we  mean  that  a  certain 
individual,  human  being  or  animal,  suffers  injury  from  the  admin- 
istration of  a  substance  which,  in  similar  amounts  and  methods  of 
administration,  does  not  exert  any  injurious  action  upon  normal 
members  of  the  same  species. 

In  some  cases,  such  as  pollen  hypersensitiveness  and  some  other 
food  idiosyncrasies,  the  hypersusceptibility  may  be  congenital,  that 
is,  inherited  by  the  individual  without  traceable  previous  contact 
with  the  substance  to  which  he  is  sensitive. 

In  most  instances,  however,  some  form  of  previous  physiological 
contact  with  the  particular  substance  seems  to  be  necessary  for  the 
development  of  the  hypersensitive  state. 

Owing  to  the  varied  manifestations  of  specific  hypersusceptibility, 
it  has  been  necessary  to  classify  the  phenomena  of  this  nature  into 
two  main  subgroups.  In  doing  this  we  follow  the  classification  of 
Doerr.1  Doerr  has  grouped  together  all  phenomena  of  hypersus- 

1  Doerr,  Kolle  and  Wassermann  Handb.,  2,  1913,  947. 

352 


HYPERSUSCEPTIBILITY  353 

ceptibility  under  the  general  term  of  allergy,  which  means  altered 
reaction.  Under  this  heading  he  classifies,  A,  those  forms  of  hyper- 
susceptibility  in  which  the  inciting  substance  is,  as  far  as  we  know 
at  the  present  time,  non-antigenic,  and,  B,  that  form  of  allergy  in 
which  the  inciting  substance  is  a  known  antigen. 

We  ourselves  would  rather  define  these  subdivisions  as,  A,  those 
forms  of  allergy  in  which  the  mechanism  of  the  hypersusceptibility 
cannot  be  shown  to  be  due  to  an  antigen-antibody  union,  and,  B,  those 
forms  in  which  an  antigen-antibody  union  within  the  body  can  be 
proved  to  be  responsible. 

The  difference  is  a  slight  one,  but  may,  in  the  future,  perhaps 
become  a  fundamental  one,  for  our  recent  studies  on  the  tuberculin 
reaction  make  us  believe  that  the  conception  of  the  word  "antigen" 
will  necessarily  change  in  the  course  of  the  next  few  years  of  in- 
vestigation. 

We  do  not  see  any  particular  purpose  in  altering  the  Doerr 
classification  to  one  suggested  by  Coca2  in  which  the  general  term 
*  *  hypersensitiveness ' '  is  subdivided  into  true  anaphylaxis  and  allergy, 
the  term  "allergy"  here  being  confined  to  the  reactions  in  which 
no  true  antigen-antibody  reaction  can  be  determined.  In  fact,  we 
believe  that  this  would  be  harmful,  in  that  Coca  thereby  implies 
that  there  is  a  general  identity  of  mechanism  underlying  the  mani- 
festations which  he  classifies  together  as  "allergy,"  some  of  which 
are  certainly  open  to  justified  differences  of  opinion. 

We  will  first  deal  with  those  forms  of  hypersusceptibility  in 
which  truly  antigenic  substances  are  involved,  and,  for  this  form, 
we  may  reserve  the  term  of  True  Anaphylaxis. 

Anaphylaxis. — As  early  as  1893,  Behring3  and  his  pupils4  had  noticed 
that  animals,  highly  immunized  against  diphtheria  toxin,  with  high  antitoxin 
content  of  the  blood,  would  occasionally  show  marked  susceptibility  to  injec- 
tions of  small  doses  of  the  toxin. 

The  phenomena  observed  by  them  was  interpreted  as  an  increased  tissue 
susceptibility  to  the  toxin,  and  Wassermann,  reasoning  on  the  basis  of 
Ehrlich's  side-chain  theory,  formulated  the  conception  that  the  increased 
susceptibility  was  due  to  toxin  receptors,  increased  in  number  by  immuniza- 

2  Coca,  Tice  ;s  System  of  Medicine,  Vol.  3,  1920. 
3Behring,  Deut.  med.  Woch.,  1893. 

4  Knorr,  Dissert.,  Marburg,  1895 ;  Behring  und  Kitashina,  Bert,  klin,  Woch., 
1901, 


354  INFECTION  AND  IMMUNITY 

tion,  but  not  yet  separated  from  the  cells  that  had  produced  them;  the  cells 
thereby  becoming  more  vulnerable  to  the  poison.  In  the  same  category 
belongs  the  observation  of  Kretz,  who  noticed  that  normal  guinea-pigs  did 
not  show  any  reaction  after  injections  of  innocuous  toxin-antitoxin  mixtures, 
but  that  marked  symptoms  of  illness  often  followed  such  injections  when 
made  into  immunized  guinea-pigs.  Other  phenomena  which  are  now  re- 
garded, a  posteriori,  as  probably  depending  upon  the  principles  involved  in 
anaphylaxis,  are  the  tuberculin  and  mallein  reactions,  fully  described  in 
another  place,  and  the  adverse  effects  often  following  the  injections  of  anti- 
toxins in  human  beings,  conditions  spoken  of  under  the  heading  of  "serum 
sickness."  The  last-named  condition  has  been  made  the  subject  of  an  exhaus- 
tive study  by  v.  Pirquet  and  Schick.5 

That  the  injection  of  diphtheria  antitoxin  in  human  beings  is  often 
followed,  after  an  incubation  time  of  from  three  to  ten  days,  by  exanthematous 
eruptions,  urticaria,  swelling  of  the  lymph  glands,  and  often  albuminuria 
and  mild  pulmonary  inflammations,  has  been  noticed  by  many  clinicians, 
who  have  made  extensive  therapeutic  use  of  antitoxin.  It  was  recognized 
early  that  such  symptoms  were  entirely  independent  of  the  antitoxic  nature 
of  the  serum,  but  appended  upon  other  constituents  or  properties  peculiar 
to  the  antitoxic  serum.  Moreover,  symptoms  of  this  description  were  by 
no  means  regular  in  patients  injected  for  the  first  time,  but  seemed  to 
depend  upon  an  individual  predisposition,  or  idiosyncrasy,  v.  Pirquet  and 
Schick,  however,  noticed  that  in  those  injected  a  second  time,  after  intervals 
of  weeks  or  months,  the  consequent  evil  effects  were  rapid  in  development, 
severe,  and  occurred  with  greater  regularity. 

The  fundamental  observations  from  which  our  present  knowledge  of 
anaphylaxis  takes  its  origin  are  those  made  in  1898  by  Hericourt  and 
Richet,6  who  observed  that  repeated  injections  of  eel  serum  into  dogs  gave 
rise  to  an  increased  susceptibility  toward  this  substance  instead  of  im- 
munizing the  dogs  against  it.  Following  up  the  lines  of  thought  suggested 
by  this  phenomenon,  Portier  and  Richet7  later  made  an  interesting  observa- 
tion while  working  with  actino-congestin — a  toxic  substance  which  they  ex- 
tracted from  the  tentacles  of  Actinia.  This  substance  in  doses  of  0.042  gram 
per  kilogram  produced  vomiting,  diarrhea,  collapse,  and  death  in  dogs.  If 
doses  considerably  smaller  than  this  were  given  in  quantities  sufficient  to 
cause  only  temporary  illness,  and  several  days  allowed  to  elapse,  a  second 
injection  of  a  quantity  less  than  one-quarter  or  one-fifth  of  the  ordinary 
lethal  dose  would  cause  rapid  and  severe  symptoms  and  often  death.  Similar 

5  Pirquet  and  Schick,  "Die  Serum  Krankheit,"  monograph,  Leipzig  and  Wien, 
1905. 

6  Hericourt  and  Ricliet,  Compt.  rend,   de  la  soc.  de  biol.,  5,3,  1898. 

7  Portier  and  Eichet,  Compt.  rend,  de  la  soc.  de  biol.,  1902  j   Eiclict,  Ann.  de 
Tinst.  Pasteur,  1907  and  1908. 


HYPERSUSCEPTIBILITY  355 

observations  were  made  soon  after  this  by  Richet  with  mytilo-congestin,  a 
toxic  substance  isolated  from  mussels.  In  these  experiments  there  remained 
little  doubt  as  to  the  fact  that  the  first  injection  had  given  rise  to  a  well- 
marked  increased  susceptibility  of  the  dogs -for  the  poison  used. 

It  was  Richet  who  first  applied  to  thistphenomenon  the  term  "anaphylaxis" 
( avd  against,  </>vAo£ts  protection),  to  distinguish  it  from  immunization  or 
prophylaxis. 

Soon  after  Richet's  earlier  experiments,  and  simultaneously  with  his  later 
work,  Arthus8  made  an  observation  which  plainly  confirmed  Richet's  observa- 
tions, though  in  a  somewhat  different  field.  The  observation  of  Arthus  is 
universally  spoken  of  as  the  "phenomenon  of  Arthus." 

He  noticed  that  the  injection  of  rabbits  with  horse  serum  ^a  substance 
in  itself  without  toxic  properties  for  normal  rabbits)  rendered  the  rabbits 
delicately  susceptible  to  a  second  injection  made  after  an  interval  of  six 
or  seven  days.  The  second  injection — even  of  small  doses — regularly  produced 
severe  symptoms  and  often  death  in  these  animals. 

An  observation  very  similar  to  that  of  Arthus  was  made  by  Theobald 
Smith9  in  1904.  Smith  observed  that  guinea-pigs  injected  with  diphtheria 
toxin-antitoxin  mixtures  in  the  course  of  antitoxin  standardization,  would  be 
killed  if  after  a  short  interval  they  were  given  a  subcutaneous  injection  of 
normal  horse  serum. 

The  fundamental  facts  of  hypersusceptibility  had  thus  been  observed, 
and  Otto,10  working  directly  upon  the  basis  of  Smith's  observation,  carried 
on  an  elaborate  inquiry  into  the  phenomenon.  Almost  simultaneously  with 
Otto's  publication  there  appeared  a  thorough  study  of  the  condition  by 
Rosenau  and  Anderson.11 

The  researches  of  Otto,  and  Rosenau  and  Anderson,  besides  confirming 
the  observations  of  previous  workers,  brought  out  a  large  number  of  new 
facts.  They  showed  conclusively  that  the  action  of  the  horse  serum  had 
no  relationship  to  its  toxin  or  to  its  antitoxin  constituents,  that  the  "sensitiza- 
tion"  of  the  guinea-pigs  by  the  first  injection  became  most  marked  after  a 
definite  incubation  time  of  about  ten  days.  Sensitization  was  accomplished 
by  extremely  small  doses  (one  one-millionth  in  one  case,  usual  doses  1/250 
to  1  c.c.).  Rosenau  and  Anderson,  furthermore,  excluded  hemolysin  or 
precipitin  action  as  explanations  of  the  phenomena,  and  proved  that  hyper- 
susceptibility  was  transmissible  from  mother  to  offspring,  and  that  it  was 
specific — animals  sensitized  with  horse  serum  not  being  sensitive  to  subsequent 


8  Arthus,  Compt.  rend,  de  la  soc.  do  biol.,  55,  1903. 
'  Th.  Smith,  Jour.  Med.  Bes.,  1904. 
111  Otto,  "Leuthold  Gedenkschrif t, "  1905. 

11  Roscnaik  and  Anderson,  Hyg.  Lab.  U.  S.  Pub.  Healt'i  and  Marine  Hosp.  Serv. 
Bull.,  29,  36,  1906,  1907. 


356  INFECTION  AND  IMMUNITY 

injections  of  other  proteins.  These  authors,  Vaughan12  and  Wheeler,  Nicolle,13 
and  others,  furthermore,  showed  that  the  reaction  was  by  no  means  limited 
to  animal  sera,  but  was  elicited  by  proteins  in  general,  pepton,  egg  albumin, 
milk,  the  extract  of  peas,  and  bacterial  extracts. 

These  observations,  together  with  those  of  many  other  workers 
which  we  must  omit  for  the  sake  of  conciseness,  were  the  funda- 
mental ones.  In  the  time  immediately  following  this  first  work, 
many  theories  of  anaphylaxis  were  advanced  and  many  faulty  ideas 
conceived,  justifiable  in  the  light  of  the  knowledge  available  at  that 
time,  but  no  longer  tenable  as  more  precise  analyses  followed.  Such, 
in  our  opinion,  are  the  earlier  ideas  of  Gay  and  Southard,14  and  that 
of  Besredka,15  both  of  which  depended  chiefly  upon  the  premise  that 
the  substance  which  sensitized  in  the  first  injection  was  not  the 
same  as  that  which  incited  the  harmful  effect  at  the  second  or  subse- 
quent injections.  Into  the  same  category  at  the  present  time  belong 
the  earlier  views  of  Wolf-Eisner,16  and  in  a  less  definite  way,  the 
theories  of  Vaughan  and  Wheeler.17  The  latter,  however,  have  had 
an  important  influence  upon  subsequent  developments  which  will  be 
referred  to  below.  Von  Pirquet  and  Schick,18  from  the  beginning, 
maintained  the  analogy  of  the  anaphylactic  phenomena  to  other 
immune  reactions,  and  believed  that  the  reaction  was  dependent 
essentially  upon  an  antigen-antibody  union;  and  similar  views  were 
held  by  Rosenau  and  Anderson,19  from  the  beginning.  In  order  to 
make  the  subject  clear,  however,  without  unnecessarily  lengthening 
its  discussion,  we  must  abandon  the  historic  method  of  treatment, 
and  define  the  various  elements  that  enter  into  the  reaction  more 
systematically. 

Wells20  has,  in  our  opinion,  most  concisely  laid  down  the  criteria 
which  must  be  met  in  the  light  of  our  present  knowledge,  in  order 
that  a  condition  may  be  regarded  as  one  of  true  anaphylaxis.  They  are, 

12  Vaughan,  Assn.  Am.  Phys.,  May,  1907. 

13  Nicolle,  Ann.  de  1'Inst.  Pasteur,  2,  1903. 

14  Gay  and  Southard,  Jour.  Med.  Ees.,  May,  1907. 

15  Besredka  and  Steinhardt,  Ann.  de  1'Inst.  Past.,  1907. 

16  Wolf-Eisner,  Berl.  klin.  Woch.,  1904. 

17  Vaughan  and  Wheeler,  Jour.  Inf.  Dis.,  4,  1907. 

18  Von  Pirquet  and  Schick,  Die  Serum  Krankheit,  Vienna,  1905. 

19  Rosenau  and  Anderson,  Hyg.  Lab.  U.  S.  Pub.  Health  and  Marine  Hosp.  Serv, 
Bull.,  29,  36,  1906  and  1907. 

?0  Wells,  Physiological  Reviews,  1,  No.  1,  January,  1921, 


HYPERSUSCEPTIBILITY  357 

with  a  few  commentaries  and  slight  modifications,  as  follows:  "The 
observed  toxicity  of  the  injected  material  must  depend  upon  sen- 
sitization  of  the  animal,  that  is,  the  substance  must  not  produce 
similar  symptoms  in  the  non-sensitized  animal"  (of  the  same 
species).  "It  should  be  possible  to  demonstrate  passive  sensitization 
with  the  serum  of  a  sensitized  animal. ' '  This  we  would  modify  some- 
what since,  of  course,  in  the  early  stages  of  developing  hypersus- 
ceptibility,  an  animal,  though  sensitive,  may  have  but  slight  or  even 
no  demonstrable  antibodies  in  his  serum.  It  would  perhaps  be  more 
accurate  to  say  that  it  must  be  possible  to  produce  passive  sensitiza- 
tion to  an  antigen  by  the  administration  of  serum  which  contains 
antibodies  to  this  antigen.  If  dealing  with  guinea  pigs,  it  should  be 
possible  by  the  Dale  Method,  to  demonstrate  typical  reactions  as 
described  below  with  the  uterus  of  the  sensitized  guinea  pig.  After 
recovery  from  anaphylactic  shock,  a  condition  of  desensitization 
should  be  apparent  if  quantitative  conditions  are  taken  into  account. 
This  is  not  all  that  Wells  says  about  it,  and  we  have  somewhat 
modified  it  for  our  own  purposes,  but,  in  general,  these  criteria  are 
the  chief  ones  which  must  be  met,  and  they  all  boil  down  to  the 
statement  that,  in  order  to  be  considered  true  aiiaphylaxis,  it  must 
be  shown  that  the  mechanism  of  whatever  reaction  that  may  occur 
is  one  that  is  fundamentally  based  upon  the  meeting  of  an  antigen 
with  its  homologous  antibody.  Further  limitations  of  this,  as  to  the 
site  and  manner  of  such  meeting  will  be  described  below. 

THE  ANAPHYLACTIC  ANTIGEN. — We  may  save  much  discussion  for 
the  purposes  of  this  particular  book  by  saying  that  substances  with 
which  true  aiiaphylaxis  can  be  produced  are  all,  as  far  as  we  know, 
protein  in  nature.  No  conclusive  proof  has  ever  been  brought  that 
lypoids  or  carbohydrates  can  act  as  antigens,  and  work  with  protein- 
split  products  has  not  been  sufficiently  satisfactory.  Racemized 
proteins  have  been  shown  by  Ten  Broeck21  not  to  exert  antigenic 
action  for  anaphylaxis.  Wells,  himself,  states  that  he  has  always 
obtained  negative  results  with  protein  cleavage  products,  but  says 
that  Fink22  in  his  own  laboratory  has  occasionally  obtained  slight 
anaphylactic  reactions  with  proteose  fractions  obtained  from 
coagulated  egg-white  by  hydrolysis  with  steam  under  pressure,  for 
those  parts  of  the  proteose  solution  which  were  precipitated  by 

21  Ten  Eroeck,  Jour.  Biol.  Chem.,  17,  1914,  369. 

22  FinTc,  Jour.  Inf.  Dis.,  25,  1919,  97. 


358  INFECTION  AND  IMMUNITY 

complete  and  three-fourths  saturation  with  ammonium  sulphate.  It 
is  interesting  to  note  that  these  two  fractions  also  incited  antibodies, 
by  which  precipitin  and  complement  fixations  could  be  obtained,  a 
matter  which,  in  this  case,  is  of  considerable  importance.  For 
further  discussion  of  other  claims  of  non-protein  anaphylactic  anti- 
gens, we  refer  the  reader  to  the  article  by  Coca  and  the  one  by 
Wells.  We  may  summarize  here  by  saying  that,  as  far  as  we  know 
at  the  present  time,  anaphylactic  antigens  differ  in  no  way  from 
other  antigens,  and  that  no  substance  at  the  present  time  has  been 
proven  to  be  an  anaphylactic  antigen  (in  the  sense  in  which  we 
define  the  term  above),  with  which  antibody  formation  in  animals 
has  failed.  In  other  words,  any  substance  that  can  incite  the  forma- 
tion of  true  antibodies  may  also  be  an  anaphylactic  antigen. 

THE  METHODS  OF  SENSITIZATION. — Experimental  sensitization.  may 
be  active  or  passive,  and  differs  to  some  extent  according  to  the 
species  of  animal  under  observation.  The  early  observations  were 
chiefly  made  on  guinea  pigs.  Guinea  pigs  can  be  actively  sensitized 
by  a  single  injection  of  various  amount.  When  dealing  with  animal 
sera  such  as  horse  serum  quantities  of  anywhere  from  0.1  to  1  c.c. 
are  most  suitable.  Minute  amounts,  however,  will  suffice,  and 
Rosenau  and  Anderson23  succeeded  in  one  case  in  sensitizing  with 
one  one-millionth  of  c.c.  of  horse  serum.  If  so  sensitized,  the  animals 
become  hypersusceptible  at  varying  periods,  hardly  ever  in  less  than 
six  days,  the  ideal  time  for  reinjection  ranging  between  two  and 
three  weeks,  somewhat  dependent  upon  the  amount  given.  Various 
statements  have  been  made  as  to  the  relationship  of  the  incubation 
time  and  the  initial  dose  given  to  guinea  pigs.  The  ideal  time  for 
injection  is  that  at  which  the  maximum  amount  of  antibody  has 
been  formed  on  the  cells  Avith  the  minimum  amounts  of  circulating 
antigen  and  antibody  in  the  blood.  The  statement  of  Coca  is  prob- 
ably right,  that,  in  general,  the  small  amounts  injected  into  guinea 
pigs  require  a  relatively  longer  incubation  period,  but  extremely 
large  amounts  (5-10  c.c.)  may  have  a  similar  effect.  The  method 
of  administration,  to  some  extent,  governs  the  incubation  period 
in  the  same  way  that  it  governs  the  speed  of  antibody  formation. 
However,  the  administration  of  the  antigen  to  guinea  pigs  and  other 
animals  may  be  carried  out  in  any  way,  except  by  feeding,  and 
even  feeding  may  result  in  a  certain  amount  of  hypersusceptibility 

23  Rosenau  and  Anderson,  loc.  cit. 


HYPERSUSCEPTIBIL1TY  359 

under  unusual  or  pathological  conditions  (abnormal  permeability  of 
the  intestinal  mucosa). 

When  sensitizing  guinea  pigs  with  bacterial  proteins  or  pollen 
and  some  other  vegetable  proteins,  it  is  necessary  to  inject  anywhere 
from  six  to  ten  times  on  consecutive  days,  and  testing  about  three 
weeks  after  the  last  injection. 

In  dogs  active  sensitization  is  hard  to  obtain  by  one  injection. 
Single  doses  of  2  to  3  c.c.  of  normal  horse  serum  are  usually  followed 
by  sensitization  in  three  weeks,  at  least  such  results  seem  to  have 
been  obtained  with  some  regularity  by  Simonds24  and  others.  Such 
sensitization,  however,  is  not  as  acute  and  severe  as  that  generally 
observed  in  guinea  pigs  under  similar  conditions.  Weil,25  however, 
obtained  acute  shock  in  dogs  by  giving  two  sensitizing  injections 
within  a  few  days,  and  testing  with  large  quantities  of  serum  after 
two  and  three  weeks.  • 

Rabbits  are  difficult  to  sensitize  with  a  single  dose  under  any 
circumstances,  but  may  easily  be  sensitized  by  repeated  injection. 

The  lower  monkeys  are  extremely  difficult  to  sensitize  under  any 
circumstances.26 

Our  knowledge  of  sensitization  in  man  is  of  course  based  entirely 
upon  clinical  observation.  And  it  is  held,  especially  by  one  inves- 
tigator of  anaphylaxis,  that  man  cannot  be  sensitized.  This,  how- 
ever, does  not  seem  to  us  to  be  tenable,  and  anaphylaxis  in  man  is 
not  an  uncommon  observation  as  manifested  by  serum  sickness,  im- 
mediate skin  reaction  and  accidents  observed  especially  among 
asthmatics  treated  with  foreign  protein  for  one  or  another  clinical 
reason.  Fatal  acute  shock  in  man,  however,  is  fortunately  rare. 

PASSIVE  SENSITIZATION. — It  is  this  phenomenon  of  passive  sensi- 
tization which  has  thrown  the  most  important  light  upon  the  process. 
It  was  first  demonstrated  by  Nicolle,27  by  Otto,28  and  by  Gay  and 
Southard,29  all  of  whom  showed  that  the  hypersusceptible  state 
could  be  passively  transferred  to  normal  animals  by  injecting  them 
with  the  serum  of  anaphylactic  animals.  In  such  experiments  the 
serum  of  the  anaphylactic  animal  is  first  injected  in  quantities  of 


24  Simon ds,  Jour.  Infcc.  Dis.,  19,  1916. 

25  Weil,  Jour.  Tmmun.,  2,  1917,  429. 

^7Ansser,  Proc.  Soc.  Exper.  Biol.  and  Med.,  18,  1920,  57. 

27  Nicolle,  Ann.  de  1  'Inst.  Past.,  2,  1903. 

28  Otto,  Munch,  med.  Woch.,  1907. 

29  Gay  and  Southard,  Jour.  Med.  Ees.,  May,  1907. 


360 


INFECTION  AND  IMMUNITY 


0.5  c.e.  or  preferably  more,  and  twenty-four  hours  later  an  injection 
of  the  specific  antigen — that  is,  the  protein  used  for  sensitization — 
is  given.  The  animals  so  treated  show  typical  symptoms  of  hyper- 
susceptibility  and  often  die. 

Simultaneous  inoculation  of  the  two  substances,  either  mixed  or 
injected  separately,  does  not  produce  the  same  effect.  A  fact, 
observed  by  Otto,  is  that  the  serum  of  guinea-pigs  who  have  been 
given  the  sensitizing  or  first  injection  will  confer  passive  anaphylaxis 
on  the  eighth  or  tenth  day  after  injection,  before  the  animals  them- 
selves show  evidences  of  being  actively  hypersensitized.  It  is  also 
true  that  occasionally  the  serum  of  antianaphylactic  animals  will 
possess  the  power  of  conferring  passive  anaphyiaxis. 

It  is  by  means  of  the  passive  method  of  sensitization  that  the 
relations  between  anaphylaxis  and  antibodies  have  been  most  suc- 
cessfully studied.  Doerr  and  Russ30  showed  that  the  power  of  a 
serum  to  convey  anaphylaxis  passively  depended  directly  upon  its 
contents  of  specific  antibody.  It  was  then'  shown  by  Nicolle,31 
Otto,32  and  others,  that  a  sharp  reaction  can  be  produced  by  this 
method  only  when  a  distinct  interval  not  less  than  four  to  six  hours, 
was  allowed  to  lapse  between  the  injection  of  the  antibodies  and 
the  injection  of  the  antigen.  This  may  be  taken  as  an  axiom  for 
all  cases  in  which  the  antigen  is  an  unformed  protein  in  solution. 

It  was  also  shown  by  Weil33  that  passive  sensitization  could  be 
conferred  by  the  injection  of  precipitates  formed  in  the  test  tube 
between  an  antiserum  and  its  antigen,  a  thing  which  we  can  now 
well  understand  in  view  of  the  knowledge  we  have  of  the  dissociation 
of  antibody  from  such  precipitates. 

Anaphylaxis  may  be  transmitted  passively  by  inheritance.  Thus 
the  young  of  anaphylactic  guinea-pigs  show  hypersusceptibility, 
irrespective  of  whether  the  mother  became  hypersusceptible  before 
or  after  the  beginning  of  pregnancy.  Such  anaphylaxis  has  no 
reference  to  the  condition  of  the  father,  and  is  not  transmitted  by 
the  milk. 

The  nature  of  these  anaphylactic  antibodies  has  aroused  much 
discussion.  By  many  observers  they  are  regarded  as  special 

80  Doerr  and  Russ,  Ztschr.  f.  Immunitatsforsch.,  1909,  iii. 
"Nicolle,  Bull,  de  PInst.  Past.,  1907,  v. 

32  Otto,   Das    Theobald   Smithsche    Phaenomenon,  .  etc.,   von    Leuthold    Gedenk- 
schrift,  1905,  i. 

33  Weil,  Jour,  of  Immunol.,  1,  1916,  19. 


HYPERSUSCEPTIBILITY  361 

anaphylactic  antibodies,  separate  from  precipitins,  opsonins,  etc., 
etc. ;  Friedberger,34  himself,  from  the  beginning,  identified  them  with 
precipitins.  The  direct  quantitative  relationship  between  pre- 
cipitating antibodies  and  the  power  to  convey  passive  sensitization. 
described  by  Doerr  and  Russ35  would  point  in  the  same  direction 
as  would  the  above  mentioned  experiments  of  Weil.  Since  we  have 
variously  expressed  in  the  preceding  pages  our  own  opinion  that 
all  antibodies  developed  against  a  single  antigen  are  one  and  the 
same  substance,  we  have  no  hesitation  in  stating  that  we  think  that 
the  so-called  sensitizing  antibody  is  identical  with  the  sensitizing 
antibody  which  is  formed  to  the  antigen,  rendering  it  amenable  to 
agglutination  or  precipitation  or  complement-fixation. 

Where  Does  the  Reaction  Occur. — As  we  have  seen,  when  the 
antigen  and  antibody  are  injected  simultaneously  or  within  a  very 
short  period  of  one  another,  no  anaphylactic  symptoms  occur.  The 
study  of  this  interval  has  gradually  led  to  the  recognition  that 
the  anaphylactic  reaction,  whatever  it  may  be,  takes  place  upon  the 
body  cells  and  that  the  interval  in  passive  sensitization  is  neces- 
sitated by  the  time  required  for  the  anchoring  of  the  antibodies 
to  the  cells  of  the  tissues.  Experiments  by  Pearce  and  Eisenbrey36 
(1910)  showed  definitely  that  a  hypersusceptible  dog  remained  sen- 
sitized even  when  his  entire  blood  volume  was  substituted  with  that 
of  a  normal  dog.  The  principle  has  been  made  especially  clear  by 
the  introduction  of  direct  methods  of  observation  of  the  smooth 
muscle  cells  of  animals,  by  Schultz37  and  by  Dale,38  a  method  which 
has  been  particularly  developed  by  Weil.39  It  seems  fairly  clear 
from  this  work  and  a  volume  of  other  researches  which  cannot  be 
reviewed  here,  that  acute  protein  anaphylaxis  as  we  see  it  in  guinea- 
pigs  and  other  laboratory  animals  is  due  to  the  direct  reaction 
between  antigen  and  a  specific  antibody  when  this  reaction  takes 
place  upon  the  body  cells  and  not  in  the  blood  stream.  Just  how 
much  influence  the  reaction  within  the  blood  stream  can  exert  or 


34  Friedberger,  with  Hartoch,  Zeit.  f .  Immunit.,  3,  1909. 

35  Doerr  and  Buss,  Zeit.  f.  Immunit.,  3,  1909. 

36  Pearce  and  Eisenbrey,  Congr.  Am.  Phys.  and  Surg.,  1910,  viii. 

37  Schultz,  Jour.  Pharmacol.  and  Exper.  Therap.,  1910,  i. 

38  Dale,  Jour.  Pharmacol.  and  Exper.  Therap.,  1913,  iv. 

39  Weil,  Jour.  Med.  Eesearch,  27,  1913;  30,  1914;  Proc.  Soc.  Exper.  Biol.  and 
Med.,  1914,  xi,  86. 


362 


INFECTION   AND   IMMUNITY 


whether  it  takes  any  important  part  in  the  phenomena  is  at  present 
a  matter  of  considerable  doubt. 

We  can  feel  safe,  therefore,  in  stating  very  definitely  that  the 
site  of  the  anaphylactic  reaction,  that  is,  the  place  at  which  the 
union  between  antigen  and  antibody  occurs  in  the  production  of 
the  various  symptoms  of  anaphylactic  injury  is  upon  the  cells.  That, 
in  other  words,  the  important  reaction  which  determines  the  train 
of  symptoms  which  we  call  anaphylaxis  occurs  when  the  antigen 
goes  into  relationship  with  antibodies  which  are  still  in  some  way 
united  to  tissue  cells,  or  are,  in  the  jargon  of  immunology,  "sessile" 
upon  the  cells. 

Whether  or  not  any  injury  may  occur  when  antigen  meets  anti- 
body in  the  circulation  is  still  an  open  question.  There  are  experi- 
ments on  record  by  Friedemann40  in  wliich  he  obtained  reactions 
in  rabbits  by  the  simultaneous  intravenous  injection  of  antigen  and 
antibody,  and  similar  occasional  occurrences  have  been  observed 
by  Brion,  Scott  and  ourselves.  But  these  reactions  are  neither 
regular  in  occurrence,  nor  are  they  ever  very  severe.  As  a  matter 
of  fact,  as  we  have  shown,  the  union  of  antigen  and  antibody  in 
the  circulation  is  inhibited  probably  by  colloidal  protection,  and 
this  may  be  regarded  as  being  very  likely  a  protective  mechanism. 
As  a  matter  of  fact,  Weil  and  others  have  shown  that  a  sufficient 
amount  of  antibody  in  the  circulation  may  even  protect  the  cells 
to  some  extent  against  anaphylactic  shock,  and  it  is  interesting 
to  note  in  this  connection  that  very  large  doses  of  protective  anti- 
serum  are  necessary  to  bring  about  this  result,  a  circumstance  which 
is  again  easily  explained  by  the  inhibition  of  union  between  cir- 
culating antigen  and  antibody. 

Concerning  the  possibility  of  the  formation  of  poisonous  sub- 
stances in  the  circulation,  produced  by  the  union  of  antigen-anti- 
body, a  question  which  is  involved  in  Friedberger's  theory  of 
anaphylaxis,  we  will  have  more  to  say  in  a  subsequent  paragraph. 

SYMPTOMS  OF  ANAPHYLAXIS. — Anaphylaxis  differs  in  its  symp- 
tomatology and  pathology  according  to  the  species  of  animal  in 
which  shock  is  produced.  There  are  certain  fundamental  systemic 
reactions  which  are  common  to  all  species,  but  in  each  species  that 
has  been  observed,  there  are  particular  localization  of  the  immediate 
and  severe  changes  which  lead  to  acute  death.  As  general  symptoms 

40  Friedemann,  Zeit.  f.  Immunit.,  2,  1909. 


HYPERSUSCEPTIBILITY  363 

we  may  enumerate  drop  in  blood  pressure,  fall  of  temperature, 
diminution  of  leucocytes,  increased  flow  of  chyle,  and  certain  meta- 
bolic disturbances,  the  identity  of  which  in  different  animal  species 
have  not  been  worked  out. 

In  guinea  pigs,  as  first  demonstrated  by  Auer  and  Lewis,  the 
typical  lung  inflation  which  leads  to  respiratory  death  is '  due  to 
spasms  of  the  muscles  of  the  bronchioles. 

In  rabbits,  acute  death  is  not  respiratory,  but  is  a  circulatory 
one,  and  has  been  shown  by  Coca41  to  be  due  to  spasms  of  the 
muscular  coats  of  the  arterioles  of  the  pulmonary  circulation,  the 
rabbit's  lung  during  shock  development  remarkably  increased  pres- 
sure against  the  passage  of  perfusion  fluid. 

In  dogs,  acute  anaphylactic  symptoms  have  been  localized  in 
the  liver  by  Manwaring42  and  others.  This  peculiar  physiological 
difference  in  various  animals  in  reaction  to  the  same  general 
mechanism  of  injury  has  been  difficult  to  understand,  but  recent 
observations  of  Coca,  Simonds,  Huber  and  Koessler43  and  others 
have  been  correlated  by  Wells  into  what  seems  to  us  a  very  rational 
and  likely  explanation.  Wells  calls  attention  to  the  fact  that  acute 
death  in  guinea  pigs  is  due  to  spasm  of  the  bronchial  muscles,  and 
that  anatomically  the  guinea  pig  has  a  very  high  development  of 
musculature  in  the  bronchii,  the  smaller  bronchioles  being  " prac- 
tically nothing  but  muscular  tubes."  Similarly,  Coca's  findings  in 
relation  to  the  pulmonary  circulation  of  rabbits  coincides  with  the 
histological  demonstration  that  the  pulmonary  arteries  of  the  rabbit 
show  a  marked  muscular  development.  Simonds  has  shown  that  the 
hepatic  veins  of  dogs  differ  from  those  of  all  other  animals  in  having 
a  highly  developed  musculature,  and  it  would  seem,  as  Wells  points 
out,  as  though  the  localization  of  acute  changes  in  different  organs 
in  the  various  animals  were  dependent  upon  fortuitous  differences 
in  the  anatomical  distribution  of  the  smooth  muscle.  He  also  points 
out,  as  further  evidence,  that  fatal  reactions  in  man  have  occurred 
only  or  most  frequently  in  persons  suffering  from  chronic  pulmonary 
conditions,  chiefly  asthma;  and  Huber  and  Koessler  have  shown 
that  asthmatic  people  develop  a  hypertrophy  of  the  bronchial  mus- 
culature which  in  its  final  histology  is  closely  analogous  to  that  of 
guinea  pigs. 

41  Coca,  Jour.  Immimol.,  4,  1919,  219. 

42  Manwaring,  Jour,  of  Immimol.,  2,  1917,  517. 
"Huber  and  Koessler,  Arch.  Int.  Med.,  1921. 


364  INFECTION  AND  IMMUNITY 

This  explanation  of  Wells  seems  to  us  eminently  logical.  We 
would  add  to  it  only  the  following  consideration.  Acute  death  may 
well  be  caused  directly  by  the  acute  spasm  of  smooth  muscle  tissue, 
and  the  acute  pathological  manifestations  may  be  dependent  upon 
the  distribution  of  such  muscle.  This  does  not,  however,  exclude 
the  likelihood  that  severe  anaphylactic  injury  may  be  caused  in 
other  cells  in  the  body  as  well,  but  these,  not  being  able  to  react 
by  acute  contraction,  or  by  any  other  pathological  alteration  that 
can  cause  acute  and  sudden  death,  may  still  be  injured  severely 
without  there  being  an  immediately  noticeable  effect. 

ANTI-ANAPHYLA.XIS. — When  sensitized  animals  recover  from 
anaphylactic  shock,  they  do  not  react  to  a  subsequent  injection  of 
the  same  substance  made  within  a  reasonable  interval. 

This  desensitization  or  ' '  antianaphylaxis ' '  as  Besredka  and 
Steinhardt  have  called  it,  appears  immediately  after  recovery  from 
the  second  injection.  Antianaphylaxis  may  also  be  produced  if 
animals  which  have  received  the  first  or  sensitizing  dose  are  injected 
with  comparatively  large  quantities  of  the  same  substance  during 
the  preanaphylactic  period — or,  as  it  is  sometimes  spoken  of,  during 
the  anaphylactic  incubation  time.  This  injection  should  not  be  done 
too  soon  after  the  first  dose,  but  rather  toward  the  middle  or  end 
of  the  preanaphylactic  period. 

If  given  within  one  or  two  days  after  the  sensitizing  injection, 
anaphylaxis  will  develop,  nevertheless.  The  desensitized  condition 
is  a  purely  transitory  state.  Besredka  and  Steinhardt  believe  that 
it  lasts  a  long  time,  while  Otto  found  guinea-pigs  immunized  in  the 
above  manner  to  lose  their  antianaphylaxis  within  three  weeks. 

It  is  not  at  all  necessary  to  actually  shock  an  animal  to  desen- 
sitize it.  The  doses  may  be  given  gradually,  either  in  small  frac- 
tions or  slowly  by  means  of  high  dilution,  as  by  the  method  of 
Friedberger,  and  gradual  desensitization  thereby  accomplished 
without  noticeable  harm..  This  is  of  great  practical  importance. 

Desensitization  in  the  ordinary  sense  probably  means  a  gradual 
saturation  of  the  sessile  antibodies  with  antigen.  It  can  be  demon- 
strated not  only  in  the  living  animal  but  upon  the  sensitive  uterus 
in  the  Dale  apparatus. 

Another  form  of  partial  protection  against  anaphylaxis,  by  the 
injection  of  large  amounts  of  specific  antiserum  has  been  mentioned 
above.  The  mechanism  of  this  is  obvious. 

Desensitization  by  injection  into  the  rectum  or  by  feeding  has 


HYPERSUSCEPTIBILITY  365 

been  accomplished  but  since  the  absorption  of  unchanged  antigen  by 
these  routes  is  ordinarily  slight,  little  hope  can  be  expected  in  this 
direction  for  practical  purposes. 

There  are  certain  forms  of  protection  against  anaphylactic  shock 
produced  by  the  injection  of  foreign  sera,  and  other  proteins  and 
processes  non-specific  as  far  as  the  particular  anaphylactic  mechan- 
ism is  concerned,  but  there  is  too  little  positive  knowledge  about 
these  to  permit  us  to  discuss  them  here. 

ANAPHYLATOXIN  THEORIES,  ETC. — Vaughan  and  Wheeler44  early 
suggested  that  anaphylaxis  might  be  a  poisoning  produced  as  fol- 
lows. Antibodies  are  formed  by  the  first  injection,  which  on  subse- 
quent reaction  with  the  antigen  administered  in  the  second  injection, 
lead  to  poisonous  protein-split  products.  A  similar  idea  was  ad- 
vanced by  Wolf-Eisner.45  Proceeding  from  this  general  concept, 
Friedberger,  whose  extensive  experimental  work  may  be  found  in 
many  articles  in  the  Zeitschrift  f.  Immunitatsforschung,  elaborated 
a  theory  of  anaphylaxis  which  may  be  summarized  in  the  following 
way:  When  the  antigen  and  antibody  meet  in  the  circulation,  the 
union  of  the  two  renders  the  antigen  amenable  to  complement  action, 
and  the  action  of  the  alexin  or  complement  upon  this  complex  splits 
off  from  it  a  poison  which  he  calls  ' '  anaphylatoxin. ' '  He  succeeded 
in  producing  poisonous  substances  in  vitro  by  treating  specific  pre- 
cipitates as  well  as  sensitized  and  unsensitized  bacteria  with  alexin. 
Injection  of  these  substances  into  guinea  pigs  caused  acute  death, 
analogous  in  symptoms  to  anaphylaxis.  Friedberger  and  Hartoch46 
showed  that  there  was  a  diminution  of  alexin  in  the  serum  of  animals 
suffering  from  acute  shock  in  the  course  both  of  active  and  of 
passively  transmitted  anaphylaxis.  He  showed  that  the  intravenous 
injection  of  substances  which  inhibited  complement  action  in  vitro, 
such  as,  for  instance  concentrated  salt  solution,  would  diminish  and 
sometimes  prevent  shock  in  sensitized  animals,  a  phenomenon,  how- 
ever, which  the  writer  with  Lieb  and  Dwyer47  showed  to  be  due 
to  diminution  of  the  irritability  of  smooth  muscle  caused  by  hyper- 
tonic  salt.  It  was  subsequently  shown,  however,  that  similar  poisons 
could  be  produced  from  boiled  as  well  as  from  normal  bacteria,  that 
they  could  be  produced  by  the  treatment  of  fresh  guinea  pig  serum 

44  Vaughan  and  Wheeler,  Jour.  Infec.  Dis.,  4,  1917. 

45  Wolf-Eisner,  Berl.  klin.  Woch.,  1904. 

48  Friedberger  and  Hartoch,  Zeit.  f.  Immunitat.,  1909,  3. 

47  Zinsser,  Lieb  and  Dwyer,  Jour.  Exper.  Biol.  and  Med.,  12,  No.  8,  1915. 


INDUCTION   AND   IMMUNITY 

with  kaolin,  barium  sulphate.  The  literature  of  this  subject  is  exten- 
sive. The  most  important  contributions  recent  have  hem  m;i<le  by 
I'.ordet,  iMoldovan  and  l)oerr,ls  and  Novy  and  DeKruiff.1''  All  of 
these  investigations  tend  to  show  that  guinea  pig  scrum  can  acquire, 
strongly  toxic  properties  when  treated  with  any  form  of  substance 
in  line  suspension,  whether  this  be  living  or  dead  bacteria,  or  pro- 
tozoa, or  indifferent  materials  such  as  agar,  kaolin,  etc.  Further- 
more, the  direct  injection  of  very  dilute  solutions  of  agar,  etc.,  into 
guinea  pigs  and  rabbits  may  cause  fatal  symptoms  closely  resembling 
anaphylaxis.  Moreover,  it  has  been  shown  by  a  number  of  workers 
that  blood  taken  from  guinea  pigs  and  rabbits,  either  defibrinatcd 
or  rent  rifuged  and  reinjected  before  the  dotting  was  complete,  could 
exert  similar  toxic  action.  We  believe  that  these  anaphylatoxin 
phenomena  are  of  very  great  importance,  that  they  represent  a 
phenomenon  dependent  upon  very  delicate  adjustment  of  the  col- 
loidal conditions  prevailing  in  the  circulating  blood,  and  demon- 
strate the  possible  dangers  accruing  from  the  disturbance  of  such 
conditions,  a  matter  which  has  been  particularly  emphasi/ed  by 
Jobling  and  .IVtersen.-1"  But,  we  do  not  believe  that  the  ana- 
phylatoxin phenomena  bear  a  direct  relationship  to  the  processes 
that  we  may  classify  under  true  anaphylaxis.  The  demonstration 
of  the  cellular  Localization  of  the  mechanism  which  causes  true 
anaphylactic  shock  has,  we  believe,  amply  demonstrated  this. 

SERUM  SICKNESS. — The  injection  of  foreign  proteins  into  human 
beings,  especially  in  the  form  of  horse  scrum  as  in  antitoxin  treat- 
ment, causes  a  train  of  symptoms  which  are  classified  together  as 
serum  sickness.  We  will  not  go  into  the  history  of  serum  sickness, 
however  interesting,  and  refer  the  reader  to  larger  works  on  the 
subject,  the  summaries  of  anaphylaxis  mentioned  above,  as  well  as 
the  book  of  Von  Pirquet  and  Schick,  "Die  Serum  Krankheit," 
Vienna,  1905.  A  peculiarity  of  this  condition  is  that  it  may  follow 
the  first  injection  of  horse  serum  or  any  other  foreign  protein,  as 
well  as  subsequent  ones. 

After  first  injection,  the  incubation  period  may  last  as  lonii  as 
twelve  days,  or  longer,  although  it  may  be  considerably  shorter  than 
this,  ('oca  states  that  in  24  to  48  per  cent  of  all  cases  the  incubation 


:in.l    />O,/T.  /.-it.    f.    ImiiMinitiit..   7,    UNO. 
« Novy  and  DeKruiff,  Jour.  A.  M.  A.,  (is,  1!>I7,  1524. 
80  Jobling  and  Petersen,  Jour.  Expci.   M,-,!.,   i«»,   11)14,  No.  5. 


HYPER8USCEPTIBILITY  367 

period  after  first  injection  is  less  than  eight  days,  while  in  about 
14  per  cent  it  is  longer  than  twelve  days. 

The  symptoms  of  serum  sickness  usually  consist  of  an  eruption 
at  the  site  of  injection,  which  ordinarily  comes  on  quite  early,  some 
time  before  any  general  eruption  is  noticed.  It  often  takes  an 
urticaria!  form,  and  becomes  general  after  several  days.  There  is 
usually  some  fever,  occasionally  albuminuria,  and  there  may  bo 
joint  manifestations.  The  joint  manifestations  are  often  of  a 
peculiar  nature,  with  slight  tenderness,  but  considerable  stiffness, 
and  very  little,  if  any,  objective  symptoms  of  the  joints,  and  we 
remember  distinctly  a  case  in  which  it  was  difficult  to  tell  whether 
or  not  the  patient,  who  had  received  tetanus  antitoxin  ten  days 
before,  was  developing  tetanus  or  not.  The  case  turned  out  to  be 
one  of  serum  sickness.  Serum  sickness  may  also  be  accompanied  by 
leucopaenia,  aild,  according  to  Weil,51  by  drops  in  blood  pressure 
and  decreased  coagulability  of  the  blood.  The  latter  manifestations, 
as  Wells  points  out,  bring  it  still  closer  to  true  anaphylaxis. 

When  a  human  bring  is  being  treated  for  the  second  time  with 
horse  serum  at  intervals  longer  than  two  or  three  weeks,  the  re- 
semblance to  true  anaphylaxis  is  still  greater,  and  as  Doerr  points 
out,  the  procedure  is  more  dangerous  and  will  vary  in  its  mani- 
festations, according  to  the  length  of  time  elapsing  between  the  two 
injections.  If  the  injections  are  not  very  much  more  than  a  month 
apart,  there  may  be,  according  to  Von  Pirquet  and  Schick,  an 
immediate  reaction,  which  takes  the  character  of  severe  serum  dis- 
ease. At  the  point  of  injection  there  may  be  swelling  and  edema, 
within  twenty-four  hours,  with  general  symptoms  such  as  those 
described  above  but  rather  more  severe,  within  one  or  two  days. 
If  the  injections  are  months  and  years  apart,  the  onset,  while  usually 
more  rapid  than  at  the  first  injection,  is  still  likely  to  occur  more 
rapidly  than  when  the  antigen  is  given  the  first  time. 

Van  Pirquet  and  Schick,  from  the  beginning,  believed  that  serum 
sickness  was  due  to  the  reaction  of  antigen  which  had  not  yet  dis- 
appeared from  the  circulation  of  the  patient  at  a  time  when  anti- 
bodies were  already  being  actively  formed. 

Opinions  as  to  whether'  scrum  sickness  is  to  be  regarded  as  true 
anaphylaxis  or  not,  seem  to  differ  at  the  present  time.  Coca  par- 
ticularly seems  to  hesitate  about  incorporating  these  phenomena  into 

ri  n  cil,  Jour.  Immimol.,  2,  1917,  399. 


368 


INFECTION  AND  IMMUNITY 


those  of  true  anaphylaxis.  We  cannot  go  into  the  controversial 
literature  in  this  place,  but  may  set  down  our  own  opinion  that 
we  think  that  the  time  interval  between  observation  of  symptoms 
and  injection,  on  first  administration,  the  speeding  up  of  symptoms 
in  cases  of  second  and  third  injections,  the  nature  of  the  symptoms, 
and  the  relationship  between  serum  sickness  and  antibody  formation 
in  the  patient,  as  pointed  out  by  C.  W.  Wells,52  as  well  as  more 
recently  by  MacKensie  and  Longcope,53  together  with  the  phenomena 
of  desensitization  of  patients,  leave  little  room  for  doubt  that  this 
peculiar  condition  is  fundamentally  of  an  anaphylactic  nature. 

It  is  not  so  easy  to  include  in  true  anaphylaxis  the  apparently 
inherited  sensitiveness  to  foreign  proteins  most  frequently  observed 
as  food  idiosyncrasies.  Whether  or  not  these  belong  into  the  cate- 
gory of  anaphylaxis,  the  future  must  reveal. 

SKIN  REACTIONS. — There  are  two  kinds  of  skin  reaction,  sone  which 
can  be  obtained,  for  instance,  in  horse  serum  sensitive  people  by  the 
injection  of  minute  quantities,  0.1  to  0.01  c.c.  of  horse  serum  intra- 
cutaneously  injected.  In  these  cases,  within  a  few  minutes  to  one- 
half  hour,  a  growing  urticarial  wheal  begins  to  appear  which  fades 
again  within  an  hour  or  longer.  This  reaction  has  been  variously 
used  to  determine  whether  or  not  patients  possessed  a  high  degree 
of  sensitiveness  just  before  the  administration  of  therapeutic  sera. 
We  have  recently  experimentally  studied  the  relationship  of  such 
immediate  skin  reactions  with  generalized  anaphylaxis  in  guinea 
pigs,  and  have  found  that  the  two  phenomena  correspond  with  con- 
siderable accuracy,  namely,  that  guinea  pigs  give  definite  immediate 
skin  reactions  during  the  periods  at  which  they  are  highly  sensitive 
to  intravenous  injections,  and  that  recovery  from  severe  anaphylactic 
shock  desensitized  them  not  only  to  general  anaphylaxis  but  to  the 
skin  reaction  as  well. 

There  is  another  form  of  skin  reaction  typified  by  the  tuberculin 
reaction  and  the  typhoidin  reaction  in  which  very  little  manifest 
change  occurs  during  the  first  two  hours,  but  in  which  after  twelve 
to  twenty-four  hours,  definite  inflammatory  swellings  occur  at  the 
point  of  injection  with  the  occasional  development  of  a  little  central 
neucrosis  and  hemorrhage,  and  true  cell  injury.  Such  reactions 
fade  only  after  three  or  four  days  or  longer,  and  are  similar  in 


62  Wells,  C.  W.,  Jour.  Infec.  Dis.,  16,  1915. 

53  MacKensie  and  Longcope,  Med.  Sec.  N.  Y.  Academy  Med.  April,  1921. 


HYPERSUSCEPTIBILI TY  369 

many  ways  to  the  toxin  skin  reactions  as  observed  in  the  Schick 
test  with  diphtheria  toxin.  The  relationships  of  these  latter  reac- 
tions to  anaphylaxis  has  been  much  questioned  and  with  justice. 
A  recent  analysis  by  ourselves  in  the  case  of  the  tuberculin  reaction 
has  shown  that  these  reactions  are  not  anaphylactic  in  the  ordinary 
sense,  but  are  brought  about  by  substances  probably  proteose  in 
nature  which  among  other  things,  differ  from  the  true  anaphylactic 
antigens  in  being  more  permeable  than  these,  and  penetrating  into 
cells.  We  refer  the  reader  to  our  article  in  the  J.  of  Exp.  Med., 
unpublished,  probably  1921. 

METHODS  OF  TESTING  AND  DESENSITIZATION  IN  INDIVIDUALS  ABOUT 
TO  BE  INJECTED  WITH  ANTITOXIC  OR  ANTIBACTERIAL  SERA. — A  consider- 
able degree  of  practical  importance  attaches  to  the  anaphylactic 
phenomena  in  connection  with  the  administration  of  sera.  When 
small  doses  of  tetanus  or  diphtheria  antitoxin  are  to  be  administered 
for  the  first  time,  it  is  generally  unnecessary  to  precede  this  with 
a  skin  test.  Although  we  should  always  advise  that  this  is  done 
in  asthmatics  or  in  people  who  have  suffered  from  chronic  coughs 
or  the  prolonged  pulmonary  inflammations  that  are  apt  to  occur  in 
children  following  measles,  influenza  or  whooping  cough.  This  is 
necessary  for  the  reasons  stated  above  and  pointed  out  by  Huber 
and  Koessler,  which  demonstrated  the  hypertrophy  of  smooth 
muscles  in  the  bronchioles  of  such  people. 

When  intravenous  serum  injections  are  to  be  made,  as  in  the 
treatment  of  pneumonia  and  meningitis,  it  is  best  to  precede  the 
injection  with  a  skin  test.  Such  tests  have  been  done  more  fre- 
quently than  anywhere  else,  we  believe,  at  the  Rockefeller  Hospital, 
where  0.02  c.c.  of  a  1 :10  dilution  of  horse  serum  is  injected  intra- 
cutaneously  with  a  similar  injetcion  of  sterile  salt  solution  as  control. 
In  unsensitive  people  the  wheal  of  the  injection  disappears  rapidly. 
But  in  sensitive  ones  it  will  begin  to  increase  after  five  or  ten 
minutes,  and  within  four  hours  may  show  a  large  red  erythematous, 
urticaria-like  area. 

In  such  cases,  careful  desensitization  should  be  practiced.  The 
precautions  to  be  taken  are  twofold.  One  consists  of  a  preliminary 
attempt  at  desensitization,  the  other  in  slow  administration  by  dilu- 
tion of  the  serum  during  the  injection  of  the  therapeutic  dose. 

The  desensitization  is  best  accomplished  by  the  method  of 
Besredka54  which  consists  in  the  gradual  injection  of  progressively 

64  Besredka,  Bull,  de  1  'Inst.  Past.,  6,  1908,  826. 


370  INFECTION  AND  IMMUNITY 

increasing  doses  of  the  antigen,  in  this  case  horse  serum,  over  an 
interval  of  a  number  of  hours.  Cole55  states  that  it  is  safe  to  begin 
with  a  subcutaneous  injection  of  0.025  c.c.  of  scrum,  and  at  one-half 
hour  intervals  thereafter,  giving  further  doses,  doubling  the  amount 
each  time.  Adverse  symptoms  should  of  course  lead  to  still  greater 
care  and  a  lengthening  of  the  interval.  If  by  this  careful  method 
finally  1  c.c.  has  been  given  at  a  dose  without  adverse  symptoms, 
0.1  c.c.  may  be  given  intravenously,  and  continued  every  one-half  hour 
with  double  the  dose.  Cole  recommends  continuing  this  careful 
procedure  until  about  25  c.c.  total  of  the  serum  has  been  given.  He 
then  waits  about  four  days,  and  gives  the  remainder  of  the  dose. 

It  stands  to  reason  that  no  absolute  rules  for  such  a  procedure 
can  be  given  and  that  after  the  general  principle  has  been  under- 
stood, the  individual  physician  must  be  guided  by  close  observation 
of  the  patient  and  familiarity  with  the  symptoms  to  be  expected. 

The  adverse  symptoms  to  be  expected  are  immediate  respiratory 
distress,  rapidity  of  the  pulse,  and  there  may  be  coughing. 

In  finally  giving  the  serum,  in  such  cases,  in  the  larger  quantities, 
it  should  be  diluted  with  sterile  salt  solution  by  at  least  one-half, 
and  may  be  so  given  by  gravity  that  the  first  10  c.c.  should  occupy 
about  ten  minutes. 

HAY  FEVER,  URTICARIA,  ETC. — The  clinical  condition  spoken  of  as 
hay  fever,  asthmatic  attacks  called  forth  by  apparent  hypersus- 
ceptibility  to  certain  kinds  of  dust,  the  emanation  of  animals,  etc., 
etc.,  cannot  at  present  be  classified  clearly  with  true  anaphylaxis 
because  of  certain  apparent  differences  from  ordinary  anaphylactic 
phenomena  which  cannot  be  ignored.  Dunbar56  at  first  regarded 
the  susceptibility  to  pollen  or  hay  fever  as  due  to  a  toxin.  This, 
however,  is  not  tenable  on  present  experimental  evidence.  The  toxin 
idea  is  not  tenable  because  of  the  fact  that  normal  insusceptible 
individuals  do  not  react  to  many  times  the  amounts  to  which  the 
hay  fever  patients  respond.  Numerous  studies  upon  the  inheritability 
of  the  hay  fever  tendency  seemed  to  show  that  a  tendency  to  hyper- 
sensitiveness  of  this  kind  may  be  hereditary,  an  observation  which 
in  the  train  of  a  number  of  other  investigators  has  recently  been 
corroborated  by  Cooke  and  Vander  Veer.57  They  seem  to  believe 


55  Cole,  Monograph  of  the  Eock  Inst.,  1917,  No.  7. 

58 Dunbar,  cited  from  Doerr,  Kolle  and  Wassermann  Hamlbiich,  Edit.  2. 

67  Coolce  and  Vander  Veer,  J.  of  Immun.  I,  1916,  201. 


HYPERSUSCEPTIBILITY  371 

' '  that  the  tendency  is  inherited  as  a  dominant  characteristic. ' '  The 
question  of  whether  or  not  hay  fever  is  a  phase  of  protein  sensitiza- 
tion,  then,  cannot  be  answered  either  affirmatively  or  negatively  at 
the  present  time.  It  is  accompanied  by  cutaneous  sensitiveness  to 
pollen  extracts  and,  according  to  Cooke  and  Vander  Veer,  subcu-. 
taneous  injections  of  such  extracts  into  sensitive  individuals  may 
produce  a  general  eruption.  On  the  basis  of  its  heredity,  Cooke  and 
Vander  Veer  and  Coca58  remove  the  hay  fever  complex  from  the 
class  of  protein  anaphylaxis.  Coca  claims  in  addition  that  pollen 
extracts  are  not  anti genie  in  the  ordinary  sense ,  of  the  word,  but 
in  this  he  is  mistaken,  since  Mrs.  Parker  in  our  laboratory  recently 
proved  that  extracts  made  by  us,  as  well  as  the  ones  used  by  Cooke 
and  Vander  Veer,  could  be  used  as  anaphylactic  antigens  if  the 
methods  of  sensitization  and  of  test  were  sufficiently  delicate. 

DRUG  IDIOSYNCRASIES. — Abnormal  sensitiveness  to  many  drugs  be- 
longing to  almost  all  chemical  classes  of  substances,  inorganic  and 
organic,  has  been  noted  for  years  by  clinicians.  Among  them  are 
morphin,  strychnin,  atropin,  salicylates,  halogens,  and  their  com- 
pounds, salvarsan,  etc.  These  substances  are  obviously  not  antigenic 
in  the  ordinary  sense.  The  hypersusceptibility  is  generally  specific 
at  least  for  the  chemical  group,  and  it  seems  to  be  a  fact  that  the 
reaction  elicited  in  the  individual  does  not  represent  exaggerated 
symptoms  of  the  physiological  effects  of  the  drugs,  but  are,  in  a 
general  way,  alike,  whatever  the  drug  used.  The  symptoms  usually 
come  on  rapidly,  within  a  few  hours  or  days,  and  consist  in  various 
kinds  of  skin  rashes,  and  fever.  In  such  cases,  where  salvarsan, 
iodin,  etc.,  preparations  have  been  used,  there  may  be  marked  and 
rapidly  developing  local  inflammatory  effects  at  the  point  of  inocula- 
tion. Drug  idiosyncrasies  cannot  be  transmitted  passively,  and,  so 
far,  no  conclusively  successful  experiments  on  artificial  hypersen- 
sitization  of  animals  with  these  substances  have  been  made.  There, 
is  only  one  exception  to  this,  namely  the  experiments  of  Swift59 
with  salvarsan  in  which  active  sensitization  of  guinea  pigs  with 
salvarsan-guinea  pig  serum  mixtures  resulted  in  apparently  success- 
ful, though  inconclusive,  results. 

There  is  no  adequate  explanation  at  the  present  time  for  drug 
idiosyncrasies.  The  most  reasonable  suggestion  which  has  been 
made,  however,  is  the  one  that  drugs  enter  into  some  form  of  com- 

58  Coca,  Tice's  Practice  of  Medicine,  Vol.  2,  1920. 

59  Swift,  J.  Exp.  .Med.  24,  1916,  373. 


372  INFECTION  AND  IMMUNITY 

bination  with  the  serum  proteins  of  the  host,  which  are  then  so 
altered  in  their  antigenic  properties  that  they  can  act  as  antibody 
inciting  or,  in  other  words,  antigenic  substances.  When  horse  serum 
or  other  animal  sera  for  instance  are  treated  with  iodin,  an  iodin- 
protein  combination  is  formed,  which  represents  an  antigenic  altera- 
tion of  the  original  serum  protein.  Animals  injected  with  such  an 
iodized  horse  serum  will  produce  antibodies  which  are,  to  some 
extent,  specific  for  this  iodin-protein  combination.  It  is  not  impos- 
sible that  this  is  the  fundamental  basis  for  drug  allergy,  but  there  are 
very  strong  arguments  against  this  assumption,  particularly  the 
failure  up  to  date  to  produce  such  conditions  uniformly  in  animals 
by  active  or  passive  sensitization.  It  would  seem  to  us  futile  at  the 
present  time  to  make  either  positive  or  negative  statements  in  this 
respect. 

ANAPHYLAXIS  IN  INFECTIOUS  DISEASE. — There  can  be  little  doubt 
about  the  fact  that  both  general  and  localized  hyper  sensitiveness 
play  an  important  role  in  infectious  disease.  Whenever  an  infection 
becomes  subacute  or  chronic,  the  body  may  become  sensitized  to  the 
coagulable  protein  in  the  bacterial  body.  This  we  have  ourselves 
shown  with  typhoid  protein,  and  with  tuberculous  guinea  pigs  and 
tuberculo-protein ;  and,  in  the  case  of  tuberculosis,  our  experiments 
done  with  the  Dale  method  corroborated  the  previous  experiments 
of  Baldwin,60  Krause61  and  others  who  worked  by  the  intravenous 
method.  Thus,  in  all  infectious  diseases  which  last  any  length  of 
time,  we  can  count  upon  anaphylactic  phenomena  to  participate  in 
the  general  symptomatological  and  pathological  picture.  This  sub- 
ject is  still  very  much  a  matter  of  experimental  investigation,  and 
a  discussion  of  it  would  carry  us  too  far  afield  in  the  present 
connection. 

Delayed  skin  reactions,  like  the  typhoidin,  tuberculin,  etc.,  reac- 
tion, are,  we  believe,  phenomena  of  specific  sypersensitiveness  to 
substances  of  a  somewhat  less  complex  molecular  structure  than  the 
proteins,  substances  which  do  not  produce  antibodies  in  the  usual 
sense;  these  substances  are  more  diffusible  than  are  the  true  pro- 
teins, can  get  inside  the  cells,  and  their  reaction  with  the  cells, 
then,  is  an  intracellular  one  in  which  the  intervention  of  sessile 
antibodies  is  not  necessary.  For  a  further  analysis  of  this  relation- 
ship and  the  experimental  basis  for  the  statements  we  make,  we  refer 
the  reader  to  our  unpublished  article  mentioned  above. 

"Baldivin,  J.  Med.  Kes.,  119,  1910. 

61  Krause,  Amer.  Eev.  Tuber.,  1,  1917,  65. 


SECTION  III 

PATHOGENIC   MICROORGANISMS 


CHAPTER   XX 

AN   INTRODUCTION   TO    THE    STUDY    OF    INFECTIOUS   DISEASES 

THE  RELATIONSHIP  OF  BACTERIOLOGY  TO  THE  CLINIC 
AND  TO  PUBLIC  HEALTH 

THE  problem  of  infectious  diseases  is  peculiar  in  that,  more  than 
any  other  branch  of  medicine,  it  calls  for  the  intimate  cooperation 
of  the  laboratory  worker,  the  clinician  and  the  sanitarian.  It  is  a 
subject  in  many  phases  of  which  problems  of  engineering  are  im- 
portant, in  which  food  production  plays  a  part,  and  to  which  the 
educational  and  sociological  agencies  of  the  community  as  a  whole 
can  contribute  very  materially.  The  eventual  suppression  of  in- 
fectious diseases  cannot  be  accomplished  by  any  one  of  the  agencies 
mentioned,  or  by  all  of  them  together  without  the  cooperation  of 
the  public  in  general. 

In  many  respects  the  study  of  infectious  diseases  is  the  most 
logical  branch  of  medicine  at  the  present  time.  In  many  of  these 
conditions  we  are  familiar  with  the  causative  agents,  we  know  the 
manner  in  which  they  gain  entrance  to  the  body,  where  they  lodge 
and  multiply,  where  and  how  they  form  their  secondary  foci,  what 
manner  of  poisons  they  produce,  and  what  reactions  they  call  forth 
in  the  living  animal  tissues.  We  can  often  isolate  the  bacteria  from 
the  bodies  of  the  sick  and  the  dead;  we  can  recognize  them  when 
we  isolate  them  in  locations  outside  the  body;  we  can  study  their 
biological  activities,  and  their  poisons  both  apart  from  the  animal 
body  and  in  animal  experimentation.  Moreover,  both  in  animals 
and  man  we  can  study  directly  the  reactions  of  the  body  to  invasion, 
and  the  agencies  by  which  the  invaders  are  destroyed. 

For  these  reasons,  the  first  requirement  for  a  thorough  clinical 

373 


374  PATHOGENIC   MICROORGANISMS 

comprehension  of  infectious  disease  as  it  occurs  in  man,  is  a  funda- 
mental biological  knowledge  of  the  bacteria  themselves,  their  actions 
upon  artificial  media,  the  characteristics  by  which  they  can  be  dif- 
ferentiated, the  conditions  under  which  they  grow  and  produce 
poisons,  and  the  reactions  which  they  or  their  poisons  elicit  in 
animals.  Of  course  there  are  many  points  in  such  a  chain  of  reason- 
ing which  investigation  has  not  yet  cleared  up,  and  there  is, 
especially,  much  uncertainty  and  half-knowledge  in  regard  to  some 
of  the  most  important  phases  of  the  chemical  and  immunological 
reactions  which  take  place  between  the  invaders  and  the  animal 
body.  Moreover,  it  is  quite  likely  that  many  of  our  theories  con- 
cerning the  fundamental  principles  which  govern  such  reactions  are 
defective.  But  without,  at  least,  a  knowledge  of  the  biological  facts 
available,  the  physician  who  is  confronted  by  a  human  infection  is 
working  very  largely  in  the  dark.  It  is  the  task  of  the  specialized 
laboratory  worker  to  prepare  this  material  and  submit  it  to  the 
clinician  so  that  we  may  use  it  in  the  premises  of  his  reasoning. 

Considered  in  this  way,  an  infection  in  an  animal  or  a  human 
being  resolves  itself  into  a  balance  between  the  forces  of  infection, 
on  the  one  hand,  and  the  injuries  and  resisting  mechanisms  of  the 
infected  subject,  on  the  other. 

We  may  consider  the  entrance  of  a  foreign  living  being  into  the 
tissues  of  a  higher  animal  or  plant  as  a  process  in  which  a  struggle 
for  existence  is  initiated.  The  invading  microorganism  must  take 
its  nourishment  from  the  invaded  host,  abstracting  thereby  materials 
needed  by  the  host,  and,  in  the  course  of  its  multiplication  and 
digestive  processes,  it  not  only  injures  the  host  mechanically  by 
local  accumulation,  but  also  by  the  remote  action  of  the  substances 
which  it  produces  in  the  course  of  its  metabolism,  many  of  which 
are  toxic.  The  host,  if  not  overwhelmed,  responds  by  reactions 
which  express  themselves  both  in  local  morphological  changes  in 
places  where  direct  contact  with  the  bacteria  is  established,  and  by 
remote  systemic  reactions  incited  by  absorption  not  only  of  the  toxic 
derivatives  of  the  bacteria,  but  also  to  some  extent  of  the  products 
of  the  local  struggle  in  which  proteolytic  destruction,  necrosis,  etc., 
are  involved.  The  symptom  complex,  therefore,  which  we  recognize 
as  disease  results  in  part  from  injury,  but  to  a  larger  extent  repre- 
sents the  manifestations  of  the  defense  reactions  of  the  host.  When 
a  physician  makes  a  diagnosis  of  a  particular  infection  by  "history 
and  physical  examination,  he  does  so  on  the  basis  of  his  own  observa- 


INTRODUCTION  TO  THE  STUDY  OF  INFECTIOUS   DISEASES     375 

tions  and  of  those  of  his  clinical  predecessors,  which  have  taught 
him  that  a  certain  kind  of  bacterial  invader  elicits  characteristic 
reactions  on  the  part  of  the  host.  In  many  cases,  however,  micro- 
organisms of  entirely  different  biological  classes  may  elicit  clinical 
pictures  of  great  similarity,  because  of  analogous  methods  of  inva- 
sion, like  degrees  of  virulence  and  similar  selective  distribution. 
Pneumonias  caused  by  pneumococci  and  Friedlander  bacilli  may 
show  very  little  clinical  difference  for  these  reasons,  and  septicemic 
invasions  of  the  blood  by  a  variety  of  microorganisms  may  not  be 
clinically  differentiable.  On  the  other  hand,  one  and  the  same 
species  of  microorganism  may  cause  widely  divergent  clinical  pic- 
tures under  conditions  of  varying  balance  between  the  virulence  of 
the  invader  and  the  resistance  of  the  host.  A  streptococcus  of  low 
virulence  in  a  vigorous  subject  may  for  instance  cause  only  a  local- 
ized abscess,  whereas,  if  the  virulence  is  enhanced  and  the  subject 
very  susceptible,  the  localized  symptoms  may  be  negligible  and  a 
septicemia  with  secondary  localizations  and  fatal  in  outcome  may 
result. 

The  accurate  diagnosis  of  an  infectious  disease,  therefore, 
depends  first  of  all  upon  a  clinical  understanding  of  the  reactions 
of  the  human  body  with  the  different  microorganisms  that  can 
invade  it,  a  knowledge  of  the  manner  in  which  the  different  forms 
can  enter  the  body,  how  they  progress  and  are  distributed,  where 
in  the  body  they  are  apt  to  accumulate,  how  great  a  degree  of  fluctua- 
tion in  virulence  and  toxicity  can  be  expected  from  them,  what 
poisons  they  produce  and  what  the  pharmacological  action  of  these 
poisons  is.  While  such  information  may  often,  and  must  frequently 
suffice  to  make  the  diagnosis,  yet  a  knowledge  of  the  manner  in 
which  blood  cultures,  urine  cultures,  stool  cultures,  throat  cultures, 
etc.,  can  be  made,  is  necessary  to  affirm  the  diagnosis  in  relatively 
clear  cases  and  to  determine  it  in  doubtful  cases;  and  this  implies 
not  only  a  knowledge  of  the  preceding  facts,  but  also  both  the 
knowledge  and  the  technical  ability  supplied  by  the  trained  bac- 
teriologist. 

Moreover,  the  dependence  of  clinical  understanding  of  infectious 
diseases  upon  bacteriological  knowledge  is  not  a  bit  less  intimate 
than  is  that  of  preventive  measures  or  sanitation. 

For  purposes  of  prevention,  every  infected  individual  or  every 
carrier  of  a  pathogenic  organism  may  be  regarded  as  the  potential 
source  for  infection  of  other  human  beings. 


376  PATHOGENIC   MICROORGANISMS 

Some  diseases  will  of  necessity  remain  sporadic  because  the  infec- 
tion of  a  new  individual  can  be  brought  about  only  by  unusually 
depressed  resistance  or  by  accidentally  enhanced  virulence  on  the 
part  of  the  causative  agent.  And  in  some  diseases  transmission 
requires  conditions  of  contact  which  are  not  an  ordinary  feature 
of  intercourse  between  the  members  of  communities.  Diseases,  thus, 
which  under  ordinary  conditions  of  civilized  life  may  occur  more 
or  less  frequently  as  sporadic  scattered  cases,  may  become  epidemic 
only  when  life  in  army  cantonments,  in  crowded  and  unsanitary 
city  quarters,  subject  to  poverty,  filth  and  neglect,  produces  com- 
munity susceptibility  and  facilitates  transmission.  Such,  as  we  shall 
see,  is  the  case  with  many  respiratory  infections,  especially  the 
pneumonias. 

Other  diseases,  on  the  other  hand,  are  characteristically  epidemic 
because  the  ordinary  virulence  of  the  microorganisms  is  such  that 
practically  all  normal  human  beings  may  be  regarded  as  susceptible 
and  because  the  most  important  avenues  of  invasion  are  open  in  the 
course  of  normal  community  association.  Such  diseases  are  plague, 
smallpox,  cholera,  influenza  and,  to  a  less  extreme  degree,  the  enteric 
fevers. 

For  this  reason,  the  ultimate  basis  of  sanitation  in  infectious 
diseases  depends  upon  close  observation  of  the  possible  sources  of 
infection  so  that  they  may  be  circumscribed  before  broadcast  dis- 
semination has  taken  place;  it  involves  the  routine  safeguarding 
of  ordinary  community  life  in  such  a  way  that  the  avenues  of  trans- 
mission for  the  various  possible  invaders  may  be  controlled,  and 
finally  it  necessitates  attention  to  the  maintenance  of  the  resistance 
of  the  community  as  a  whole,  both  by  the  hygiene  of  every  day  life, 
the  prevention  of  undue  lowering  of  resistance  of  large  groups  by 
economic  or  other  hardships,  or,  as  in  smallpox  and  typhoid  fever, 
by  the  artificial  reenforcement  of  community  resistance  by  methods 
of  immunization. 

The  source  of  infection  lies  invariably  in  direct  or  indirect  trans- 
mission of  microorganisms  from  a  human  or  animal  source.  Every 
case  of  infection  represents  the  possible  origin  of  many  others,  and 
necessitates  surrounding  the  patient  with  all  the  safeguards  appro- 
priate to  the  type  of  infection  from  which  he  is  suffering,  based 
on  knowledge  of  the  manner  in  which  the  particular  disease  is 
transmitted.  If  the  diseased  individual  were  the  only  problem,  the 
task  would  be  relatively  easy.  As  we  shall  see,  however,  in  the 


INTRODUCTION  TO  THE  STUDY  OF   INFECTIOUS   DISEASES     377 

subsequent  chapters,  a  great  many  infectious  agents  may  live  sapro- 
phytic  lives  in  and  upon  the  bodies  of  human  beings,  who  themselves 
are  not  suffering  from  the  disease.  Such  individuals  are  known  as 
carriers,  and  the  carrier  problem  has  infused  difficulties  into  sanitary 
procedure  which,  in  some  cases,  it  is  almost  impossible  to  combat. 
Thus,  every  community  has  in  it  a  definite  percentage  of  typhoid 
and  paratyphoid  carriers;  many  individuals  carry  meningococci, 
diphtheria  bacilli  and  virulent  pneumococci  and  streptococci;  there 
are  quite  surely  carriers  of  poliomyelitis  and  scarlet  fever,  and  it 
is  not  impossible  that  the  carrier  state  can  exist  for  a  number  of 
other  diseases  in  which  we  have  not  yet  been  able  to  prove  the 
condition  by  actual  experiment. 

In  addition  to  the  case  and  the  carrier,  a  source  of  great  danger 
are  unrecognized  mild  cases.  Typhoid  fever  may  take  a  very  mild 
form,  especially  in  vaccinated  people;  the  atypical  cases  of 
poliomyelitis  which  occur  in  the  course  of  every  epidemic  and  may 
occur  in  interepidemic  periods  may  not  be  recognized  until  secondary 
cases  occur;  and  in  many  adults  who  possess  a  relatively  high  im- 
munity, diphtheria  infection  of  the  throat  may  be  so  mild  that  no 
suspicion  of  the  disease  is  aroused.  It  is  in  connection  with  such 
occurrences  that  the  diagnostic  acumen  of  the  practicing  physician 
is  especially  important,  and  it  is  in  the  recognition  of  the  atypical 
cases  and  in  the  early  diagnosis  of  the  ordinary  cases,  that  the 
practitioner  represents  the  first  line  of  defense  against  epidemic 
outbreaks. 

It  is  for  this  part  of  the  protective  campaign  that  we  need 
reporting  systems,  so  that  early  cases  may  be  centrally  collected  and 
charted.  Organizations  for  epidemiological  survey  to  trace  the  early 
cases  to  their  sources,  and  laboratory  units  to  affirm  the  diagnosis, 
search  out  the  possible  carriers  and  trace  the  infection,  if  possible, 
to  contaminated  food  or  water.  Here,  too,  are  necessary  arrange- 
ments for  isolation  and  hospitalization  which  involve  the  bac- 
teriological control  by  which  may  be  determined  when  it  is  safe  to 
release  the  patient  for  free  association  with  his  fellows. 

The  transmission  of  infectious  disease  may  be  either  by  direct 
contact  from  person  to  person,  by  indirect  contact  through  materials 
that  have  passed  from  the  sick  or  the  carrier  to  the  new  victim 
by  food  and  water  and  by  conveyance  through  the  agency  of  insects. 
These  factors  will  be  considered  in  detail  in  connection  with  every 
individual  disease,  and  it  is  quite  clear  that,  in  order  to  properly 


378  PATHOGENIC   MICROORGANISMS 

safeguard  a  community,  it  is  necessary  to  know  where  in  the  body 
of  the  sick  or  the  carrier  the  organisms  are  to  be  found;  how  they 
may  leave  the  body;  what  the  viability  of  the  infectious  agent  is  in 
nature;  how  long  it  can  live  under  different  conditions  of  environ- 
ment in  the  interval  between  its  leaving  one  body  and  entering 
the  next,  and  by  which  channels  it  most  easily  infects.  Also,  the 
habits  of  insects  that  can  carry  disease  must  be  studied. 

To  interrupt  the  chain  of  transmission  from  source  to  victim, 
.also,  there  must  be  a  routine  organization  for  the  safeguarding  of 
water,  food  and  other  agencies  which  in  crowded  communities  are 
always  in  danger  of  contamination.  And  in  special  cases  it  may 
involve  emergency  measures  of  personal  hygiene,  engineering  prob- 
lems and  insect  destruction. 

In  thinking  of  infectious  diseases  from  the  point  of  view  of 
sanitation  it  is  well  to  carry  them  in  one's  mind  in  the  tentative 
classification  based  upon  means  of  transmission,  since  for  each  par- 
ticular subdivision  of  this  kind,  preventive  measures  will  fall  into 
a  definite  common  plan  of  procedure. 

Thought  of  in  this  way,  all  the  infectious  diseases  fall  into  four 
groups : 

(1)  The  first  of  these  consists  of  those  transmitted  by  the  respira- 
tory channels.  This  means  that  the  infectious  virus  leaves  the  case, 
convalescent  or  carrier,  witli  the  saliva  or  mucus  of  the  upper 
respiratory  passages  and  enters  the  new  victim  by  the  same  chan- 
nels. On  this  basis,  the  respiratory  group  would  consist  in  fhe 
common  cold,  the  pneumonias,  influenza,  scarlet  fever,  measles, 
smallpox,  chickeiipox,  mumps,  whooping  cough,  tuberculosis,  diph- 
theria, meningitis,  poliomyletis  and  a  few  others. 

In  dealing  with  such  diseases,  the  task  is  to  suppress  conditions 
which  would  make  it  possible  for  a  wholesale  distribution  of  sputum, 
for  close  contact  between  individuals  in  sleeping  quarters,  the 
avoidance  of  crowding  in  homes,  schools,  institutions,  etc.,  ventila- 
tion, dust  prevention,  and,  in  short,  to  diminish  as  much  as  possible 
the  opportunities  for  close  individual  contact  in  closed  spaces.  In 
this  group  of  diseases,  prevention  is  most  difficult  because  it  is  quite 
obvious  that  contacts  need  no1  be  of  long  duration,  and  that  close 
association  in  public  vehicles,  and  in  the  ordinary  course  of  business 
and  social  intercourse  may  suffice  for  transmission.  In  these  dis- 
eases, particularly,  when  there  is  universal  susceptibility,  as  in  the 
case  of  influenza  or  smallpox,  or  some  of  the  exanthemata  in  the 


INTRODUCTION  TO  THE  STUDY  OF   INFECTIOUS  DISEASES     379 

case  of  children,  epidemic  spread  is  almost  unavoidable  if  unsani- 
tary, close  association  exists.  Fortunately,  in  many  of  the  diseases 
so  transmitted,  namely,  pneumonia,  meningitis,  diphtheria  and  a 
number  of  others,  the  normal  resistance .  of  the  human  being  is 
relatively  high  and  epidemic  occurrence  takes  place  only  when 
unusual  conditions  prevail. 

(2)  The  intestinal  group  consisting  very  largely  of  typhoid  and 
the  paratyphoid  fevers,  the  dysenteries,  cholera,  the  food  poisonings, 
and  the  simple  diarrheas.  In  this  group  sanitation,  apart  from  isola- 
tion of  the  recognized  case,  focuses  upon  a  constant  vigilance  in 
regard  to  the  distribution  of  dejecta  from  human  beings,  for  every 
infection  signifies  a  sanitary  defect  in  the  transmission  of  the  con- 
tents of  the  bowels  of  one  human  being  to  the  mouth  of  another  by 
any  one  of  a  variety  of  routes.  Sanitation  here  requires  proper 
sewage  disposal,  the  care  of  privies  and  latrines,  and  careful  control 
of  water  and  food  supplies,  since  naturally  large  epidemics  can  be 
most  easily  brought  about  in  this  group  by  the  contamination  of 
the  daily  diet  of  the  community.  In  these  diseases,  wholesale  infec- 
tion with  water  and  milk  is  constantly  diminishing,  as  we  are  im- 
proving in  our  sanitary  organizations.  But  contact  epidemics  from 
person  to  person  becoming  relatively  more  prominent.  Kecent 
studies  are  tending,  for  instance,  to  show  more  and  more  the  in- 
creasing importance  of  contact  infection  in  typhoid  fever.  Schule1 
studying  the  reports  of  the  German  Laboratory  at  Trier  for  1918, 
states  that  of  the  typhoid  cases  occurring  in  the  district  controlled 
by  this  laboratory  during  that  year,  60  per  cent  were  due  to  contact 
with  cases,  5  per  cent  were  due  to  contact  with  carriers  and  only 
1  per  cent  were  due  to  water  and  milk  each.  Of  5,889  cases  analyzed 
at  this  laboratory,  71  per  cent  were  contact  cases. 

The  manner  of  contact  may  vary,  and  Gay2  summarizes  in  the 
following  way,  the  manner  in  which  this  may  take  place : 

1.  Fingers  or  utensils — mouth. 

2.  Fingers — food — mouth. 

3.  Fomites — fingers — food — mouth. 

4.  Flies — food — mouth. 

5.  Fingers — flies — food — mouth. 

In  the  case  of  the  first  route,  more  or  less  direct  contact  with 
a  case  is  implied.  In  each  of  the  five  routes  it  is  easy  to  draw 

1  Schule,  Mil.  Surgeon,  45,  1919,  268. 

2  Gay,  Typhoid  Fever,  Macmillan  Company,  N.  Y.,  1918. 


380  PATHOGENIC   MICROORGANISMS 

vertical  lines  across  the  dashes  and  see  where  and  how  sanitary 
interference  may  interrupt  the  progress  of  transmission.  Thus,  in 
the  prevention  of  the  intestinal  infections,  the  municipal  and  state 
authorities  must  care  for  the  water  supplies  and  sewage  disposal 
plants ;  the  administrative  public  health  authorities  must  be  supplied 
with  a  reporting  system  for  the  early  recognition  and  demarkation 
of  foci,  and  must  epidemiologically  attempt  to  trace  existing  cases 
to  their  sources,  following  this  with  carrier  examinations  of  sus- 
pected small  groups ;  physicians  must  act  as  the  first  line  of  defense 
in  regard  to  early  diagnosis,  intelligent,  immediate  isolation  and  the 
collection  of  the  first  significant  epidemiological  information  for  the 
use  of  the  health  authorities;  city  cleaning  departments  and  other 
agencies  must  aid  in  preventing  fly  breeding,  in  garbage  disposal, 
etc.,  and  last  but  not  least,  general  educational  campaigns  must 
elicit  the  intelligent  cooperation  of  the  public  by  supplying  the 
simple  information  which  is  necessary  for  individual  protection.  As 
a  matter  of  fact,  if  the  public  realized  that  most  cases  of  typhoid 
and  the  paratyphoid  fevers  could  be  suppressed  at  the  present  time 
by  a  regular  hand  washing  after  defecation  and  before  meals,  the 
problem  would  be  largely  solved. 

(3)  The  third  group  comprises  diseases  in  which  intimate  contact 
between  infectious  material  and  the  external  surface  of  the  body 
of  the  new  victim  is  necessary.     In  some  of  these  diseases  trans- 
mission can  take  place  without  a  visible  break  in  the  skin,  as  perhaps 
in  plague,  rabies,  syphilis  and  some  others,  where  the  lesion  through 
which  the  virus  can  enter  may  be  so  microscopical  in  size  that  no 
visible  trauma  is  apparent.     In  others,  such  as  the  pyogenic  infec- 
tions,  glanders,   anthrax  and  the  anaerobic  infections,  trauma  of 
some  kind  is  usually  necessary.     Few  of  these  diseases  can  ever 
become  epidemic  to  any  great  extent  in  the  ordinary  sense  of  the 
word.     Some  of  them,  however,  like  the  venereal  diseases  may  be 
regarded  as  so  plentifully  endemic,  owing  to  the  nature  of  their 
transmission,  that  we  may  look  upon  the  present  condition  of  com- 
munities as  subject  to  a  constant  subacute  epidemic  state.    Venereal 
diseases   are   a   special   sanitary   group   which  requires   individual 
treatment  which  we  cannot  enlarge  upon  in  this  place. 

(4)  In  the  fourth  group  are  those  diseases  which  are  transmitted 
by  insects.    Such  are  malaria,  yellow  fever  and  dengue  fever,  trans- 
mitted by  different  species  of  mosquitoes,  African  sleeping  sickness, 
pappataci  fever,  transmitted  by  flies,  Rocky  Mountain  spotted  fever 


INTRODUCTION  TO  THE  STUDY  OF  INFECTIOUS  DISEASES      381 

and  African  relapsing  fever,  transmitted  by  a  species  of  tick, 
European  and  Balkan  relapsing  fever,  and  kala-azar  transmitted  by 
bed  bugs,  and  typhus  fever,  trench  fever  and  some  varieties  of 
relapsing  fever  transmitted  by  lice,  and  plague  largely  transmitted 
by  fleas. 

In  the  sanitation  of  these  diseases  it  is  quite  obvious  that,  in 
addition  to  our  knowledge  of  the  pathogenic  microorganisms  which 
cause  the  disease,  we  must  study  carefully  the  habits  of  insects, 
their  seasonal  occurrence,  their  nocturnal  or  diurnal  habits,  their 
methods  and  places  of  breeding,  the  distances  which  they  can  travel, 
the  manner  in  which  they  acquire  the  parasites  and  the  manner  in 
which  they  can  be  suppressed. 

Based  upon  an  understanding  of  the  conditions  outlined  above, 
moreover,  is  the  science  of  epidemiology  which,  in  its  turn,  can  in- 
directly contribute  a  great  deal,  both  to  the  solution  of  the  problems 
of  bacteriology  and  to  those  of  transmission.  For,  by  epidemiological 
surveys,  by  a  careful  study  of  the  manner  of  spread  of  disease 
from  group  to  group,  and  from  place  to  place,  the  explosiveness 
with  which  it  appears  and  relationship  of  cases  to  personal  contact, 
water  or  milk  supplies,  etc.,  many  deductions  can  be  made  which 
have  important  bearing  in  guiding  bacteriological  investigation  and 
preventive  measures.  As  to  preventive  measures,  nothing  is  so  im- 
portant in  the  suppression  of  an  epidemic  disease  as  the  rapid 
circumscription  of  the  initial  focus,  a  thing  which  can  be  accom- 
plished only  by  prompt  epidemiological  survey,  backed  up  by 
accurate  bacteriological  diagnosis.  Such  results  can  be  achieved 
only  when  communities  have  efficient  organizations  for  the  prompt 
reporting  of  communicable  diseases,  for  the  systematic  charting  of 
the  cases  and  for  intelligent  and  experienced  study  of  such  charts. 
Indeed,  studies  of  this  nature  alone  may  often  make  possible  a 
causal  classification  of  the  epidemic  before  its  bacteriological  nature 
is  accurately  known.  For  epidemics  will  vary  in  the  respects  men- 
tioned above,  very  largely  according  to  whether  they  are  water 
borne,  distributed  by  milk  or  other  food,  or  whether  they  are  spread 
by  contact  or  by  insects. 

It  is  probable  that  small  water  epidemics  from  individual  rural 
house  supplies  may  still  be  quite  frequent  and  simulate  contact 
epidemics.  The  larger  water  epidemics  of  typhoid,  cholera,  etc.,  will 
become,  as  we  have  stated,  more  and  more  infrequent  as  water 
supplies  are  more  carefully  supervised,  but  when  they  do  occur, 


382  PATHOGENIC   MICROORGANISMS 

their  onsets  and  courses  will  be  characteristic  to  a  degree  which 
makes  it  possible  for  an  experienced  epidemiologist  to  suspect  the 
water  simply  by  a  study  of  the  incidence  of  cases  in  time  and  place. 
To  some  extent,  of  course,  this  depends  upon  whether  the  water 
is  polluted  by  a  single  introduction  of  large  quantities  of  sewage  or 
whether  contamination  is  continuous  over  a  longer  period.  In  both 
cases,  however,  a  large  number  of  the  people  living  in  the  area 
of  the  water  supply  will  be  infected  during  a  relatively  short  period 
of  time.  The  rise  of  the  curve  which  can  be  constructed  from  the 
cases,  therefore,  will  be  steep  and  rapidly  reach  a  peak.  Classical 
instances  of  this  are  the  cholera  epidemic  in  Hamburg  and  an 
epidemic  of  typhoid  fever  in  an  American  city  which  is  described 
in  the  section  on  typhoid  fever.  The  incidence  of  the  cases  in  places 
will  be  sharply  limited  by  the  distribution  of  the  water  supply  and 
examination  of  the  water  will  reveal  colon  bacilli. 

In  milk  epidemics,  a  still  more  explosive  rise  of  the  cases  will 
appear  and  in  such  cases  as  the  Stamford  epidemic  described  by 
Trask,  a  definite  connection  between  the  milk  route  and  the  dis- 
tribution of  cases  may  be  traced.  On  the  basis  of  such  suspicion, 
the  bacteriologist  can  investigate  the  milk  and  attempt  a  determina- 
tion of  recent  intestinal  disease  or  the  carrier  state  in  milk  handlers. 
Also,  it  is  stated  by  many  who  have  studied  these  epidemics  that 
milk  epidemics  are  apt  to  claim  the  largest  numbers  of  victims 
among  women  and  children. 

Contact  epidemics  will  proceed  by  a  more  insidious  course.  In 
such  epidemics  it  is  extremely  important  to  gather  together  careful 
data  concerning  the  earliest  cases  observed  and  to  attempt  to  trace 
the  cases  to  some  association  at  a  common  meal,  a  restaurant  or  at 
some  other  common  source  of  food.  Sawyer  traced  a  "contact 
epidemic  to  the  carrier  state  in  a  ship's  cook  by  a  simple 
epidemiological  study  of  the  individual  cases  which  all  led  by 
separate  trails  to  the  same  ship 's  galley.  We  have,  ourselves,  traced 
cases  in  this  way  to  company  kitchens  in  military  units.  In  large 
communities  contact  epidemics  may  trail  along  for  long  periods  of 
time  and  when  association  is  indiscriminate  it  may  be  necessarily 
impossible  to  establish  accurate  relationships.  In  some  contact 
epidemics,  such  as  those  occurring  in  the  Allied  Armies  in  France 
when  intestinal  diseases  appeared  in  large  numbers,  the  rise  of  the 
curve  simulated  that  of  a  water  epidemic  very  largely  because  the 
indiscriminate  distribution  of  unprotected  dejecta  over  large  areas 


INTRODUCTION  TO  THE  STUDY  OF  INFECTIOUS  DISEASES      383 

of  recently  occupied  territory,  the  prevalence  of  flies,  and  the  crowd- 
ing of  large  masses  of  men  in  small  areas  made  frequent  contact 
unavoidable.  Such  conditions,  however,  are  not  likely  to  happen 
to  any  extent  at  ordinary  times,  and  contact  epidemics  are  usually 
limited  in  area  and  distribution,  and  careful  epidemiological  study 
of  three  or  four  cases  may  lead  to  the  original  focus  which  can  then 
be  determined  by  prompt  laboratory  investigation. 


CHAPTER   XXI 

THE  STAPHYLOCOCGI   (MICROCOCCI) 

THE  power  to  incite  purulent  and  sero-purulent  inflammations  and 
localized  abscesses  in  man  and  animals  is  possessed  by  a  large  variety 
of  pathogenic  bacteria.  Most  infections,  in  fact,  in  which  the  rela- 
tive virulence  of  the  incitant  and  the  resistance  of  the  infected 
subject  are  so  balanced  that  temporary  or  permanent  localization 
of  the  infectious  process  takes  place  are  apt  to  be  accompanied  by 
the  formation  of  pus.  The  large  majority  of  acute  and  subacute 
purulent  processes,  however,  are  caused  by  the  members  of  a  well- 
defined  group  of  bacteria  spoken  of  as  the  pyogenic  cocci.  Among 
these,  pre-eminent  in  importance,  are  the  " staphylococci"  or  "micro- 
cocci." 

Many  of  the  earlier  investigators  of  surgical  infections  had  seen 
small  round  bodies  in  the  pus  discharged  from  abscesses  and  sinuses 
and  had  given  them  a  variety  of  names.  Careful  bacteriological 
studies,  however,  were  not  made  until  1879  and  the  years  imme- 
diately following,  when  Koch,  Pasteur,  Ogston,1  and  others  not  only 
described  morphologically,  but  cultivated  the  cocci  from  surgical 
lesions  of  animals  and  man.  Of  fundamental  importance  are  the 
studies  published  by  Rosenbach2  in  1884,  in  which  the  technical 
methods  of  modern  bacteriology  were  brought  to  bear  upon  this  sub- 
ject for  the  first  time.  The  group  of  staphylococci — so  named  from 
their  growth  in  irregular,  grape-like  clusters — is  made  up  of  several 
members,  by  far  the  most  important  of  which,  pathologically,  is  the 
Staphylococcus  pyogenes  aureus. 

STAPHYLOCOCCUS  PYOGENES  AUREUS 

Morphology  and  Staining. — This  microorganism,  the  most  fre- 
quent cause  of  abscesses,  boils,  and  many  surgical  suppurations,  is 
a  spherical  coccus  having  an  average  diameter  of  about  0.8  micra, 

1  Ogston,  Brit.  Med.  Jour.,  1881. 

2  Rosenl)acli,  f '  Microorganismen  bei  Wundinf ektion, "   1884. 

384 


STAPHYLOCOCCUS  PYOGENES  AUREUS         385 

but  varying  within  the  extreme  limits  of  0.4  to  1.2  miera.  Any 
considerable  variation  from  the  average  size,  however,  is  rare.  The 
perfectly  spherical  character  may  not  develop,  whenever,  as  is 
usually  the  case,  two  or  more  are  grouped  together,  unseparated 
after  cell  cleavage.  In  this  case,  adjacent  cocci  are  slightly  flattened 
along  their  contiguous  surfaces. 

Examined  in  smears  from  cultures  or  pus,  the  staphylococci  may 
appear  as  single  individuals,  in  pairs,  or,  most  frequently,  in  irregular 
grape-like  clusters,  Occasionally,  short  chains  of  three  or  four  may 


FIG.  43. — STAPHYLOCOCCUS  PYOGENES  AUREUS.     (After  Gunther.) 

be  seen.  In  very  young  cultures  in  fluid  media,  the  diplococcus  form 
may  predominate. 

The  staphylococci  stain  with  all  the  usual  basic  aqueous  anilin 
dyes,  and,  less  intensely,  with  some  of  the  acid  dyes.  Stained  by 
the  method  of  Gram,  they  retain  the  amlin-gentian-violet.  Gram's 
method  of  staining  is  excellently  adapted  for  demonstration  of  these 
cocci  in  tissue  sections. 

Although  exhibiting  marked  Brownian  movements  in  the  hang- 
ing drop,  staphylococci  are  non-motile  and  possess  no  flagella.  They 
are  non-sporogenous  and  form  no  capsules. 

Cultural  Characters. — Staphylococci  grow  readily  upon  the  usual 
laboratory  media.  The  simpler  media,  made  of  meat  extract,  are 
quite  as  efficient  for  their  cultivation  as  are  the  freshly  made  meat- 


386  PATHOGENIC  MICROORGANISMS 

infusion  products.  The  optimum  temperature  for  staphylococcus 
cultivation  lies  at  or  about  35°  C.,  though  growth  readily  takes  place 
at  temperatures  as  low  as  15°  C.,  and  as  high  as  40°  C.  Slow  but 
denfiite  growth  has  been  observed  at  a  temperature  as  low  as  10°  C. 

While  development  is  most  characteristic  and  luxuriant  under 
aerobic  conditions,  staphylococci  are  facultatively  anaerobic  on  suit- 
able media.  They  grow  readily  in  an  atmosphere  of  hydrogen. 

As  to  the  reaction  of  media,  staphylococcus  develops  most  favor- 
ably upon  those  having  a  slightly  alkalin  titer.  Moderately  in- 
creased alkalinity  or  even  moderate  acidity  of  media  does  not  inhibit 
growth. 

On  gelatin  plates,  growth  occurs  readily  at  room  temperature, 
forming  within  thirty-six  to  forty-eight  hours,  small,  shining,  pin-head 
shaped  colonies,  appearing,  at  first,  grayish-white,  and  later  assuming 
a  yellowish  hue,  which  intensifies  into  a  light  brown  and  often  a  bronze 
color  as  the  colony  grows  older.  The  intensity  of  the  color  differs  con- 
siderably in  different  races  of  staphylococci.  Liquefaction  of  the 
gelatin  occurs,  and,  shallow,  saucer-shaped  depressions  are  formed 
about  the  colonies  after  forty-eight  hours  or  more.  These  zones  of 
fiuidification  grow  larger  as  the  colonies  grow,  finally  becoming  con- 
fluent. Microscopically,  the  colonies  themselves,  before  liquefaction 
has  destroyed  their  outline,  are  round,  rather  finely  granular,  with 
smooth  edges.  They  are  not  flat,  but  rise  from  the  surface  of  the 
medium  as  the  segment  of  a  sphere.  In  gelatin  stab  cultures  in  tubes, 
fluidification  leads  to  the  formation  of  a  funnel-shaped  depression, 
with,  finally,  complete  liquefaction  of  the  medium  and  sedimentation 
of  the  bacteria.  Liquefaction  of  gelatin  by  the  staphylococcus  is  due 
to  a  ferment-like  body  elaborated  by  it,  which  is  spoken  of  as  "gela- 
tinasQ. ' '  This  substance  can  •  be  obtained  apart  from  the  cocci  by 
the  filtration  of  cultures.3  It  is  an  extremely  thermolabile  body. 

On  agar  plates  the  characteristics  of  the  growth,  barring  liquefac- 
tion, are  much  like  those  on  gelatin.  Colonies  do  not  show  a  tendency 
toward  confluence,  remaining  discrete,  and  show  a  rather  remarkable 
difference  in  the  size  of  the  colonies  occurring  upon  the  same  plate. 
Upon  slanted  agar  in  tubes,  rapid  growth  occurs,  at  first  grayish-white, 
but  soon  covering  the  surface  of  the  slant  as  a  glistening,  golden-brown 
layer. 

In  broth,  growth  is  rapid,  leading  to  a  general,  even  clouding  of 


*Loeb,  Cent.  f.  Bakt.,  xxxii,  1902. 


STAPHYLOCOCCUS  PYOGENES  AUREUS 


387 


the  medium,  and  giving  rise,  after  forty-eight  or  more  hours,  to  the 
formation  of  a  thin  surface  pellicle.  As  growth  increases,  the  bacteria 
sink  to  the  bottom,  forming  a  heavy,  mucoid  sediment.  The  odor  of 
old  cultures  is  often  peculiarly  acrid,  not  unlike  weak  butyric  acid. 


FIG.  44. — STAPHYLOCOCCUS  COLONIES. 


In  milk,  staphylococcus  causes  coagulation  usually  within  three  or 
four  days,  with  the  formation  of  lactic  and  butyric  acids. 

On  potato,  growth  is  abundant,  rather  dry  and  usually  deeply 
pigmented. 

Upon  coagulated  animal  sera,  rapid  growth  takes  place  and  even- 
tually slight  liquefaction  of  the  medium  occurs. 

In  nitrate  solutions,  reduction  of  the  nitrates  to  nitrites  is  caused. 

In  Dunham's  broth,  indol  is  not  formed.  Bayne- Jones  and  Zin- 
ninger  have  studied  115  strains  of  various  staphylococci  in  all  kinds 
of  media  suitable  for  the  production  of  indol,  and  have  not  found  a 
single  indol  producer. 

In  media  containing  the  carbohydrates — dextrose,  lactose,  or  sac- 
charose— acidification  takes  place  with  the  formation  chiefly  of  lac- 
tic, butyric,  and  formic  acids.  There  is  no  gas  formation,  however. 


388  PATHOGENIC  MICROORGANISMS 

In  protein  media  free  from  sugars,  the  staphylococcus  produces 
alkali. 

The  reducing  action  of  staphylococcus  is  shown  by  decolorization 
in  cultures  of  litmus,  methylene-blue,  and  rosanilin.4 

Pigment  Formation. — Differentiation  between  the  various  mem- 
bers of  the  staphylococcus  group  is  based  largely  upon  the  formation 
of  pigments.  These  pigments,  so  far  as  we  know,  seem  to  be  species 
characteristics.  Thus,  Staphylococcus  pyogenes  aureus  is  recognized 
primarily  by  its  production  of  a  yellowish-brown  pigment,  varying 
in  different  strains  from  a  pale  brown  hue  to  a  deep  golden  yellow. 
Prolonged  cultivation  upon  artificial  media  may  lead  to  a  diminution 
in  the  depth  of  color  produced.5  It  appears  only  when  cultivation  is 
carried  on  under  freely  aerobic  conditions,  anaerobic  cultivation 
resulting  in  unpigmented  colonies.  The  coloring  matter  is  insoluble 
in  water  but  soluble  in  alcohol,  chloroform,  ether,  and  benzol.6 
According  to  Sch'neider,7  the  pigment  belongs  to  the  class  of  "lipo- 
chromes"  of  fatty  pigments,  and  is  probably  composed  of  carbon, 
oxygen,  and  hydrogen,  without  nitrogen.  Treatment  with  concen- 
trated sulphuric  acid  changes  it  to  a  green  or  greenish-blue.8 
Neisser9  states  that  the  pigment  of  staphylococci  is  excreted  into  the 
media  by  the  organisms  but  does  not  diffuse  because  it  is  not  soluble 
in  water.  Differences  in  pigment  have  been  the  basis  of  differentia- 
tions within  the  micrococcus  group  as  we  shall  see  below. 

Resistance. — Although  not  spore  formers,  staphylococci  are  more 
resistant  to  heat  than  many  other  purely  vegetative  forms.  The 
thermal  death  point  given  for  Staphylococcus  pyo genes  aureus  by 
Sternberg10  lies  between  56°  and  58°  C.,  the  time  of  exposure  being 
ten  minutes.  The  same  author  states  that,  when  in  a  completely 
dried  state,  the  coccus  is  still  more  resistant,  a  temperature  of  from 
90°  to  100°  C.  being  required  for  its  destruction.  Against  low  tem- 
peratures, staphylococci  are  extremely  resistant,  repeated  freezing 
often  failing  to  sterilize  cultures. 

Desiccation  is  usually  well  borne,  staphylococci  remaining  alive 

4Fr.  Muller,  Cent.  f.  Bakt.,  xxvi,  1899. 

5  Fliigge,  ' '  Die  Microorg., ' '  etc. 

*Migula,  tl System  d.  Bakt.,"  Jena,  1897. 

7  Sclmeider,  Arb.  a.  d.  bakt.  Inst,,  Karlsruhe,   1,  vol.  i,  1894. 

s Fischer,  "Vorles.  iiber  die  Bakt,,"  Jena,  190,°,. 

9  Neisser,  Kolle  and   Wassermann,  2nd  Ed.  vol.  4,  p.  369. 

10 Sternberg,  "Textbook,"  etc.,  N.  Y.,  1901,  p.  375. 


STAPHYLOCOCCUS  PYOGENES  AUREUS         380 

for  six  to  fourteen  weeks  when  dried  upon  paper  or  cloth.11  On 
slant  agar,  staphylococci  may  be  safely  left  for  three  or  four  months 
without  transplantation,  and  remain  alive.12 

The  resistance  of  staphylococci  to  chemicals,  a  question  of  great 
surgical  importance,  has  been  made  the  subject  of  extensive  re- 
searches, notably  by  Liibbert,13  Abbott,14  Franzott,15  and  many 
others.  According  to  Liibbert,  inhibition  of  staphylococcus  growth 
is  attained  by  the  use  of  boric  acid  1  in  327,  salicylic  acid  1  in  650, 
corrosive  sublimate  1  in  80,000,  carbolic  acid  1  in  800,  thymol  1  in 
11,000.  Staphylococci  are  killed  by  corrosive  sublimate  1  in  1,000 
in  ten  minutes,  by  carbolic  acid  1  per  cent  in  35  minutes,  3  per  cent 
in  2  minutes  (Franzott).  Ethyl  alcohol,16  even  when  absolute,  is 
not  very  efficient  as  a  disinfectant.  Nascent  iodin,  as  split  off  from 
iodof orm  in  wounds,  is  extremely  powerful  in .  destroying  staphy- 
lococci. 

Pathogenicity. — Separate  strains  of  Staphylococcus  pyo genes 
aureus  show  wide  variations  in  relative  virulence.  The  most  highly 
virulent  are  usually  those  recently  isolated  from  human  suppurative 
lesions,  but  no  definite  rule  can  be  formulated  in  this  respect.  The 
virulence  of  a  given  strain,  furthermore,  may  be  occasionally  en- 
hanced by  repeated  passages  through  the  body  of  a  susceptible 
animal.  Prolonged  cultivation  upon  artificial  media  is  liable  to 
decrease  the  virulence  of  any  given  strain,  though  this  is  not 
regularly  the  case.  There  are,  moreover,  unquestionably,  many 
staphylococci  constantly  present  in  the  air,  dust,  and  water,  which 
although  morphologically  and  culturally  not  unlike  the  pathogenic- 
ally  important  species,  may  be  regarded  as  harmless  saprophytes. 

The  susceptibility  of  animals  to  staphylococcus  infection  is,  like- 
wise, subject  to  extreme  variations,  depending  both  upon  differences 
between  species  and  upon  fortuitous  individual  differences  in  sus- 
ceptibility among  animals  within  the  same  species.  Animals  on  the 
whole  are  less  susceptible  to  staphylococcus  than  is  man.  Among 
the  ordinary  laboratory  animals,  rabbits  are  most  susceptible  to  this 
microorganism.  Mice,  and  especially  the  white  Japanese  mice,  show 

11  Deslong champs,  Paris,  1897. 
12Passet,  Fort.  d.  Med.,  2  and  3,  1885. 
"Liibbert,  "Bipl.  Untersuch.,"  Wurzburg,  1886. 
14  Abbott,  Medical  News,  Phila.,  1886. 
™  Franzott,  Zeit.  f.  Hyg.,  1893. 
™Hanel,  Beit.  z.  klin.  Chir.,  xxvi. 


390  PATHOGENIC   MICROORGANISMS 

considerable  susceptibility.     Guinea-pigs  possess  a  relatively  higher 
resistance.17 

Subcutaneous  or  intramuscular  inoculation  of  a  susceptible 
animal  usually  results  in  the  formation  of  a  localized  abscess  with 
much  pus  formation  and  eventual  recovery.  Intraperitoneal  inocula- 
tion is  more  often  fatal.  Intravenous  inoculation  of  doses  of  0.5 
c.c.,  or  more,  of  fresh  broth  cultures  of  virulent  staphylococci  usually 
leads  to  pyemia  with  the  production  of  secondary  abscesses,  located 
chiefly  in  the  kidneys  and  the  heart  and  voluntary  muscles,  but  not 
infrequently  in  other  organs  as  well.  In  the  kidney  they  occur  as 
small  foci,  situated  most  often  in  the  cortex,  composed  of  a  central, 
necrotic  pus  cavity,  surrounded  by  a  zone  of  acute  inflammatory 
exudation.  Staphylococcus  lesions  form  histologically  the  typical 
" acute  abscess."  Not  infrequently  the  pyemic  condition  is  accom- 
panied by  suppurative  lesions  in  the  joints.  Intravenous  injections 
of  virulent  staphylococci  preceded  by  injury  to  a  bone  is  often 
followed  by  the  development  of  osteomyelitis.  Mechanical  or  chem- 
ical injury  of  the  heart  valves  preceding  intravascular  staphylo- 
coccus  inoculation  may  result  in  localization  of  the  infection  on  or 
about  the  heart  valves,  leading  to  "malignant  endocarditis."  In 
producing  experimental  lesions  in  rabbits  all  varieties  of  staphylo- 
coccus  infection  may  be  obtained  by  suitable  methods  of  injection. 
If,  for  instance,  a  rabbit  is  given  one-half  to  1  c.c.  of  a  young  broth 
culture,  from  which  the  clumps  have  been  gently  centrifuged  down, 
into  the  ear  vein,  a  rapid  fatal  septicemia  will  result  with  organisms 
in  the  heart's  blood,  but  no  secondary  localization  or  abscess  forma- 
tion. If,  however,  Staphylococcus  cultures  containing  clumps  are 
gently  centrifuged,  the  supernatant  fluid  taken  off,  and  small  clumps 
injected  in  not  too  large  amounts  (and  the  amounts  must  be  adjusted 
to  the  virulence  of  the  culture)  the  animal  will  pass  through  a 
protracted  illness,  with  secondary  abscess  formation  in  kidneys, 
liver  and  other  organs,  in  which  emboli  have  been  formed,  a  condi- 
tion simulating  accurately  pyemia  in  human  beings.  The  pyemic 
conditions  following  Staphylococcus  inoculation  usually  lead  to 
chronic  emaciation  and  death  after  an  interval  dependent  upon  the 
relative  virulence  of  the  microorganism,  the  amount  injected,  and  the 
resistance  of  the  infected  subject. 


17  Terin,    Kef.    in    Lubarsch    und    Ostertag,    Ergebnisse,     1896 ;    Lingelsheim, 
Aetiol.  d.  Staph.  Inf.,"  etc.,  Wien,  1900. 


{STAPHYLOCOCCUS  PYOGENES  AUREUS  391 

As  above  stated,  the  susceptibility  of  man  to  spontaneous  staphy- 
lococcus  infection  is  decidedly  more  marked  than  is  that  of  animals. 
The  form  of  infection  most  frequently  observed  is  the  common 
boil  or  furuncle.  As  Garre,18  Biidinger,19  Schimmelbusch,-0  and  others 
have  demonstrated  by  experiments  upon  their  own  bodies,  energetic 
rubbing  of  the  skin  with  virulent  staphylococcus  cultures  may  often 
be  followed  by  the  development  of  a  furuncle.  Subcutaneous  inocu- 
lation of  the  human  subject  invariably  gives  rise  to  an  abscess. 
The  organisms  are  apparently  present  on  the  skin  of  human  beings 
with  great  frequency,  and  it  is  not  unlikely  that  in  the  course  of 
daily  life,  they  may  be  rubbed  into  hair  follicles  and  sweat  glands, 
and  be  present  constantly  on  some  part  of  the  body,  prepared  for 
immediate  invasion  of  an  abrasion  or  other  accident  furnishes  the 
opportunity.  A  simple  and  frequent  disease,  furunculosis,  is,  never- 
theless, a  condition  about  the  pathogenesis  of  which  we  are  con- 
siderably in  the  dark.  Reductions  of  general  resistance,  especially 
those  accompanying  overwork,  indoor  occupations,  and  faulty  diet, 
seem  to  be  concerned  in  furnishing  the  proper  conditions  for  invasion 
by  the  ever  present  staphylococci.  General  metabolic  diseases,  such 
as  nephritis  and  especially  diabetes,  render  the  individual  abnormally 
susceptible.  In  certain  instances  it  has  been  suggested,  especially 
by  Wright,  that  reduction  in  coagulation  time  of  the  blood  might 
influence  this  state  of  affairs. 

Staphylococcus  lesions  of  the  skin  are  characteristic  in  that, 
after  an  induration,  there  occurs  a  central  softening  with  the  forma- 
tion of  liquid  pus.  It  is  an  important  observation,  confirmed  by 
much  experience,  that  if  incision  is  practiced  in  the  indurated  and 
inflamed  tissue  before  the  process  has  come  to  a  central  head, 
infection  is  usually  spread,  perhaps  by  the  opening  of  adjacent 
lymphatics.  Therefore,  there  is  much  judgment  required  in  treating 
even  these  simple  lesions.  Faulty  surgical  interference  may  easily 
convert  a  simple  furuncle  into  a  dangerous  carbuncle. 

Common  among  staphylococcus  skin  infections  is  paronychia,  or 
infection  of  the  nail  bed  on  the  fingers.  This  may  often  lead  to 
troublesome  extension  up  the  fingers  and  into  the  hands. 


18  Garre,  Beit,  z.  klin.  Chir.,  x,  1893. 

w  Biidinger,  Lubarsch  und  Ostertag,  Ergebnisse,  etc,,  1896, 

20  Schimmelbusch,  Ref .  by  Biidinger. 


392  PATHOGENIC   MICROORGANISMS 

Especially  dangerous  are  boils  about  the  nose  and  lips,  and  not 
infrequently  infections  in  these  locations  may  extend  rapidly,  and 
cause  fatal  septicemia. 

Impetigo  contagiosum,  a  skin  disease  consisting  of  boil-like  in- 
flamed papules  and  occurring  particularly  in  young  children,  is 
caused  by  staphylococci. 

In  suppurative  lesions  of  the  bones,  or  osteomyelitis,  staphylococci 
are  the  most  frequent  causative  agents.  This  may  result,  after 
compound  fracture,  by  infection  from  without,  or  not  infrequently 
staphylococci  will  lodge  in  the  site  of  mechanical  injury  of  bone 
or  fracture,  reaching  the  focus  through  the  circulation.  The  lesions 
produced  in  bone  may  consist  of  slow  localized  abscesses,  or  may 
extend  along  the  medullary  canal  of  the  entire  bone. 

In  addition  to  these  most  common  lesions,  staphylococci  may 
cause  abscesses  in  almost  any  part  of  the  body.  In  cases  in  which 
resistance  if  low  and  the  staphylococci  particularly  virulent,  sep- 
ticemia may  follow  in  any  of  these.  Unlike  the  rapid,  acute  sep- 
ticemia death,  however,  which  is  likely  to  ensue  when  similar  general 
infection  with  streptococci  takes  place,  staphylococcus  generalization 
is  apt  to  lead  to  secondary  foci  in  kidneys,  liver  and  other  organs. 
This  leads  to  the  condition  of  pyemia  in  which  an  irregular  septic 
temperature  with  frequent  chills  are  characteristic.  Blood  culture 
in  such  cases  will  give  a  clue  to  the  nature  of  the  infection. 

Ascending  infections  of  the  genito-urinary  tract,  cystitis  and 
pyelonephritis,  may  be  caused  by  staphylococci. 

Staphylococcus  empyema  and  peritonitis  are  not  particularly 
common,  but  may  occur. 

Puerperal  sepsis,  while  not  as  commonly  a  staphylococcus  infec- 
tion, as  it  is  a  streptococcus  lesion,  may  occur. 

By  some  writers  staphylococci  have  been  held  responsible  for 
rheumatism,  but  there  is  no  convincing  evidence  of  this. 

Staphylococci  may  also  appear  in  meningitis.  It  is  a  curioi 
fact  that  occasionally  a  very  low  grade  staphylococcus  may  get  int< 
the  meninges,  and  cause  a  very  slow  and  apparently  mild  meningitis. 
We  have  seen  one  such  case  caused  by  a  staphylococcus  albus  recover, 
and  another  which  died  after  a  prolonged  illness  in  which  the 
organisms  were  repeatedly  isolated  from  the  spinal  fluid,  and,  at 
autopsy,  in  which  the  origin  was  a  cerebellar  abscess. 

Prolonged  chronic  infection  with  staphylococci  may  give  rise  to 
the  so-called  amyloid  changes  in  liver,  spleen  and  kidneys. 


STAPHYLOCOCCUS  PYOGENES  AUREUS         393 

Toxic  Products. — Endotoxins. — The  dead  bodies  of  staphylococci 
injected  into  animals  may  occasionally  give  rise  to  abscess  formation, 
and,21  if  in  sufficient  quantity,  may  cause  death.  To  obtain  the  latter 
result,  however,  large  quantities  are  necessary,  the  endotoxic  sub- 
stances within  the  dead  cell  body  of  these  microorganisms  being  prob- 
ably neither  very  poisonous  nor  abundant.22 

That  dead  cultures  of  Staphylococcus  aureus  exert  a  strong  posi- 
tive chemotaxis  for  leucocytes  was  shown  beyond  question  by  the 
experiments  of  Borissow.23 

Hemolysins. — In  1900  Kraus24  noticed  the  hemolytic  action  of 
staphylococci  growing  upon  blood-agar  plate  cultures.  Neisser  and 
Wechsberg25  then  showed  that  this  hemolytic  substance,  secreted  by 
the  Staphylococcus,  could  be  demonstrated  in  filtrates  of  bouillon  cul- 
tures. Such  hemolysins  are  produced  by  Staphylococcus  aureus,  and, 
to  a  lesser  degree,  by  Staphylococcus  albus.  The  quantity  produced 
varies  enormously  with  different  strains  and  seems  to  be  roughly 
proportionate  to  the  virulence  of  the  particular  microorganism,  though 
exceptions  to  this  rule  are  not  uncommon.  Absolutely  avirulent  races 
do  not,  so  far  as  we  know,  produce  hemolysins.  Relationship  of 
hemolysin  formation  to  virulence,  however,  is  not  by  any  means  as 
regular  as  at  first  supposed.  A  great  many  staphylococci  may  be 
isolated  from  human  lesions  which  produce  absolutely  no  hemolysin. 
The  culture  medium  most  favorable  to  the  formation  of  these  sub- 
stances is,  according  to  Neisser  and  Wechsberg,  a  moderately  alkalin 
beef  bouillon.  Cultivated  at  37.5°  C.,  the  bouillon  contains  the  maxi- 
mum amount  of  hemolytic  substance  between  the  eighth  and  four- 
teenth day,  and  this  may  be  separated  from  the  bacteria  by  filtration 
through  Berkefeld  or  Chamberland  filters.  We  have  not  ourselves 
attempted  to  confirm  these  investigations,  but  in  the  particular  respect 
of  lateness  of  concentration  in  the  cultures  the  Staphylococcus  hemoly- 
sin seems  to  differ  distinctly  from  that  produced  by  streptococci,  in 
which  the  optimum  for  a  large  yield  is  early,  within  the  first  twelve 
or  fourteen  hours  of  cultivation. 

The  hemolytic  action  may  be  observed  by  the  general  technique 
for  determining  hemolysis  (given  on  page  311).  It  is  important  to 

21  Scltattenfroh,  Arch.  f.  Hyg.,  xxxi,  1887. 

22  v.  Lingelslieim,  "Aetiol.  u.  Therap.  d.  Staph.  Krank.,"  Wien,  1900. 

23  Borissow,  Zieglers  Beitr.,  xvi,  1894. 

24  Kraus,  Wien.  klin.  Woch.,  iii,  1900. 

25  Neisser  und  Wechsberg,  Zeit.  f .  Hyg.,  xxxvi,  1901. 


394  PATHOGENIC   MICROORGANISMS 

wash  the  red  blood  corpuscles  used  for  the  experiments,  since  many 
animals  normally  possess  small  quantities  of  antihemolysin  in  their 
blood-sera  (man  and  horse  especially).26  The  red  blood  corpuscles  of 
rabbits,  dogs,  and  guinea-pigs  are  extremely  susceptible  to  the  action 
of  the  staphylo-hemolysin.  Those  of  man  are  less  easily  injured  by  it. 
The  hemolytic  action  takes  place,  as  Todd27  and  others28  have  shown, 
not  only  in  vitro,  but  in  the  living  animal  as  well. 

The  staphylo-hemolysin  is  comparatively  thermolabile.  According 
to  Neisser  and  Wechsberg,  heating  it  to  56°  C.  for  twenty  minutes 
destroys  it.  According  to  some  other  authors,  however,  higher  tem- 
peratures (60°  to  80°  C.)  are  required.  Reactivation  of  a  destroyed 
staphylo-hemolysin  has  so  far  been  unsuccessful.  The  fact  that  anti- 
staphylolysin  is  occasionally  present  in  normal  sera  has  been  men- 
tioned above.  This  antibody  is  most  abundant  in  the  blood  of  horses 
and  of  man.  Artificially  antistaphylolysin  formation  is  easily  induced 
by  subcutaneous  inoculation  of  staphylolysin  into  rabbits. 

Leucocidin. — In  1894,  Van  de  Velde29  discovered  that  the  pleural 
exudate  of  rabbits  following  the  injection  of  virulent  staphylococci, 
showed  marked  evidences  of  leucocyte  destruction.  He  was  subse- 
quently able  to  show  that  the  substance  causing  the  death  and  partial 
solution  of  the  leucocytes  was  a  soluble  toxin  formed  by  the  staphylo- 
coccus,  not  only  in  vivo,  but  in  vitro  as  well ;  for  cultures  of  Staphylo- 
coccus  pyogenes  aureus,  grown  in  mixtures  of  bouillon  and  blood 
serum,  contained,  within  forty-eight  hours,  marked  quantities  of  this 
' '  leucocidin. ' ' 

Other  workers  since  Van  de  Velde  have  evolved  various  methods 
for  obtaining  potent  leucocidin.  Bail30  obtained  it  by  growing  virulent 
staphylococcus  in  mixtures  of  one-per-cent  glycerin  solutions  and  rab- 
bit serum.  Neisser  and  Wechsberg31  advise  the  use  of  a  carefully 
titrated  alkalin  bouillon.  To  obtain  the  leucocidin  free  from  bacteria, 
the  cultures  are  passed  through  Chamberland  or  Berkefeld  filters, 
after  about  eight  to  eleven  days'  growth  at  37°  C.,  at  which  time  the 
contents  in  leucocidin  are  usually  at  their  highest  point. 

The  action  of  leucocidin  upon  leucocytes  may  be  observed  in  vivo 

26  Neisser,  Deut.  med.  Woch.,  1900. 

27  Todd,  Trans.  London  Path.  Soc.,  1902. 
2sKraus,  Wien.  klin.  Woch.,  1902. 

29  Van  de  Velde,  La  Cellule,  x,  1894. 

80  Bail,  Arch.  f.  Hyg.,  xxxii,  1898. 

31  Neisser  und  Wechsberg,  Zeit.  f.  Hyg.,  xxxvi,  1901. 


STAPHYLOCOCCUS  PYOGENES  AUREUS         395 

by  the  simple  method  of  Van  de  Velde,  of  injecting  virulent  staphylo- 
cocci  intrapleurally  into  rabbits  and  examining  the  exudate.  Bail 
advises  the  production  of  leucocytic  intrapleural  exudates  by  the  use 
of  aleuronat  and  following  this  after  twenty-four  hours  by  an  injection 
of  leucocidin-filtrate.  In  vitro  the  phenomenon  may  be  observed  by 
direct  examination  of  mixtures  of  leucocytes  and  leucocidin  in  the 
hanging  drop  on  a  warmed  stage,  or  by  the  t '  methylene-blue 
method"  of  Neisser  and  Wechsberg.  This  method  is  based  upon  the 
fact  that  living  leucocytes  will  reduce  methylene-blue  solutions  and 
render  them  colorless,  while  dead  leucocytes  have  lost  this  power. 
Leucocidin  and  leucocytes  are  allowed  to  remain  in  contact  for  a 
given  time  and  to  them  is  then  added  dilute  solution  of  methylene- 
blue.  If  the  leucocytes  have  been  actively  attacked  by  leucocidin, 
no  reduction  takes  place.  This  method  is  particularly  adapted  for 
quantitative  tests. 

All  staphylococcus  strains  do  not  produce  leucocidin  to  the  same 
degree.  Almost  all  true  Staphylococcus  pyogenes  aureus  cultures 
produce  some  of  this  taxin,  but  one  strain  may  produce  fifty-  and  a 
hundred-fold  the  quantity  produced  by  another.  Staphylococcus 
pyogenes  albus  gives  rise  to  this  substance  but  rarely,  and  then  in 
small  quantity. 

Leucocidin  seems  to  be  similar  to  the  soluble  toxins  of  other 
bacteria.  It  is  rapidly  destroyed  by  heat  at  58°  C.,  and  deteriorates 
quickly  in  culture  fluids  at  incubator  temperatures.  It  is  distinct 
from  staphylohemolysin  as  shown  by  differences  in  thermostability. 

Soon  after  Van  de  Velde 's  discovery  of  leucocidin,  Denys  and 
Van  de  Velde32  produced  an  antileucocidin  by  treating  rabbits  with 
pleural  exudate  containing  leucocidin.  Neisser  and  Wechsberg33 
later  confirmed  these  results  and  showed  that  among  staphylococci, 
leucocidin  is  not  specific,  the  toxin  of  all  strains  of  Staphylococcus 
aureus  and  albus  examined  being  neutralizable  by  the  same  anti- 
leucocidin. Antileucocidin  is  often  found  in  the  normal  sera  of 
horses  and  man.34 

Leucocidin  should  not  be  confounded  with  "leucotoxin,"  a  sub- 
stance obtained  in  serum  by  treatment  of  animals  with  leucocytes, 
a  true  "cytotoxin,"  having  no  connection  whatever  with  the  staphy- 
lococcus. 


32  Denys  et  Van  de  Velde,  La  Cellule,  xi,  1895. 

33Loc.  cit. 

34  Van  de  Velde,  Presse  medicale,  i,  1900. 


396  PATHOGENIC  MICROORGANISMS 

Staphylococci,  besides  the  toxic  substances  already  mentioned, 
give  rise  to  gelatinase,  spoken  of  in  the  section  upon  cultivation, 
and  to  a  proteolytic  ferment  by  means  of  which  albuminous  media 
(Loeffler's  serum)  may  be  slightly  digested. 

Immunization. — Animals  can  be  rendered  actively  immuix  by 
repeated  inoculations  with  carefully  graded  doses  of  living  or  d< -ad 
staphylococcus  cultures.35  The  production  of  antistaphylolysin  and 
of  antileucocidin  in  the  sera  of  animals  so  treated,  has  b< •< -n  allud< •<[ 
to  in  the  preceding  sections.  The  sera  of  such  actively  immuni/'  <1 
animals  possess  distinct  protective  power  when  adminisi<  n-4  to 
other  animals,  slightly  before  or  at  the  same  time  with  an  inoculation 
of  staphylococci.  They  do  not,  however,  exhibit  very  high  bacU-ri- 
cidal  power  in  vitro,  the  protective  properties  depending  probably 
upon  their  opsonic  contents.36 

Agglutinins  have  been  demonstrated  in  staphylococcus  immune 
sera  by  a  number  of  authors,  and  have  been  of  some  slight  valu<  in 
differentiating  between  the  several  groups  of  staphylococc-i.  7  A 
rather  surprising  result  of  these  researches  has  been  the  recognition 
that  immune  sera  obtained  with  pathogenic  staphylococci  will  ag- 
glutinate other  pathogenic  staphylococci,  whether  belonging  to  tin- 
group  of  Staphylococcus  pyogenes  aureus  or  that  of  Staphylocoocus 
pyogenes  albus,  but  will  not  agglutinate  any  of  the  non-pathogenic 
members  of  either  group.38  In  view  of  the  recent  studios  on  the 
antigenic  classification  of  streptococci  and  pneumococci,  a  re- 
examination  of  these  relationships  within  the  staphylococcus  group 
should  be  undertaken.  It  is  more  than  likely  that  this  group  is  a 
heterogeneous  one,  and  that,  for  purposes  of  intelligent  expM -irm -nta- 
tion  in  serum  and  vaccine  therapy,  an  antigenic  classification  should 
be  attempted. 

Active  immunization  of  human  beings  suffering  from  staphylo- 
coccus infections  has  been  extensively  practiced  by  Wright,  in  con- 
nection with  his  work  on  opsonins.  There  can  be  no  question  about 
the  fact  that  the  opsonic  substances  in  the  blood  are  increased  by 
the  injection  of  dead  staphylococci.  The  procedure  is  of  therapeutic 
value  in  subacute  and  chronic  cases.  The  work  of  Hiss  on  the  use 

*Bichet  et  Hericourt,  Compt.  rend,  de  1'acad.  des  sci.,  cvii,  1888. 
*Kolle  und  Otto,  Zeit.  f.  Hyg.,  xli,  1902. 

"  Proscher,  Cent.  f.  Bakt.,  xxxiv,  1903;  v.  Lingelsheim,  "AetioL  u.  Therap.  d. 
Staphyl.,"  etc.,  Wien,  1900. 

"Proscher,  Deut.  med.  Woch.,  xi,  1903. 


STAPHYLOC :<>(•('  I  IS    I'YO( !!  INKS   AURKIIS 

of  leucocyte  extracts  in  animals  infected  with  Staphylococcus 
pyogenes  aureus  lias  given  encouragement  for  such  treatment  in 
human  beings.  A  number  of  staphylococcus  infections  in  man  have, 
been  successfully  treated  with  leucocyte  extracts  by  Hiss  and 
Zinsser. 

Passive  immunization  with  anti-staphylococcus  sera  has  not  been 
a  therapeutic  success.  Kx  tensive  work  has  been  done  especially 
by  German  investigators  in  animal  experimentation,  but  in  most 
cases  it  lias  been  found  that  serum  injections  were  of  little  use  if 
administered  after  the.  infection  had  been  established.  Injection 
into  the  test  animals  before  infection  or  at  the  same  time  with 
infection  sometimes  gave  favorable  results.  In  man,  passive  im- 
munization has  not  been  encouraging,  although  we  believe  that  hope 
of  some  benefit  in  this  direction  should  not  be  completely  abandoned 
until  the  antigcnie  classification  of  the  staphylocoeci  has  been 
attempted. 

Hooker4"  has  recently  reported  a  number  of  cases  in  which  he 
lias  transfused  into  patients  suffering  from  staphylococcus  scp- 
ticemia  the  blood  of  donors  irnmuni/ed  with  staphylococcus  vaccines. 
This  procedure  of  course  would  be  applicable  only  to  subacute  cases, 
but  in  the  procedure  of  Hooker  there  seems  to  be  promise. 

STAPHYLOCOCCUS  PYOOKNKB  ALDUS. — This  coccus  differs  from 
Staphylococeus  pyogenes  aun-us  simply  in  the  absence  of  the  golden 
yellow  coloration  of  its  cultures.  Morphologically,  culturally,  and 
pathogenically,  it  is  in  every  way  identical  with  the  staphylococcus 
described  in  the  preceding  section,  but  its  toxin-  and  enzyme-produc- 
ing powers  in  general  are  less  developed  than  those  of  the  aureus 
variety.  Its  close  biological  relationship  to  aureus  is  furthermore 
demonstrated  by  its  agglutination  in  Staphylocoeeus  pyogenes  aureus 
immune  sera. 

ST.M'jjyi.oc'"-'  Dfi  KPIDKKMIDIS  ALBTS. — The  Staphylococcus  epi- 
dermidis  albus  described  by  Welch  is  merely  one  of  the  rion- 
mthogemc  varieties  of  Staphylococcus  pyogenes  albus  and  possibly 
loes  not  deserve  separate  classification.  It  may  give  rise  to  unim- 
portant abscesses. 

ST.\KFJYJ.or<»r  CUfi  PYDOENHE  On  BEO& — Staphylococcus  pyog<-n«-s 
'•it IT-US  produces  a  bright  yejlow  or  lemon-colored  pigment  of  dis- 
tinctly different  hue  from  that  of  Staphyloeoeeus  pyogenes  aureus. 

"  Hooker,  An.  of  Surgery,  Nov.,  1917,  p.  51.'i. 


398 


PATHOGENIC   MICROORGANISMS 


It  may  be  pyogenic  and  in  every  way  similar  to  Staphylococcus 
pyogenes  aureus,  but  is  less  often  found  in  connection  with 
pathological  lesions  than  either  of  the  preceding  staphylococci. 

A  large  number  of  staphylococci,  differing  from  those  described 
above  in  one  or  another  detail,  have  bejen  observed.  They  are  of 
common  occurrence  and  are  met  with  chiefly  as  contaminations  in 
the  course  of  bacteriological  work.  Few  of  these  have  any  patholog- 
ical significance  and  none  of  them  are  toxin-producers,  so  far  as  we 
know.  Many  of  them  differ,  furthermore,  in  their  inability  to 
liquefy  gelatin.  All  of  them  belong  more  strictly  to  the  field  of 
botany  than  to  that  of  pathological  bacteriology. 

Atypical  pathogenic  staphylococci  have  been  described  by  a  num- 
ber of  observers.  Thus  Weichselbaum40  isolated  a  Staphylococcus 
from  a  case  of  malignant  endocarditis  which  could  not  be  cultivated 
at  room  temperature,  and  grew  only  in  very  delicate  colonies.  Veil- 
Ion,41  moreover,  has  described  a  strictly  anaerobic  Staphylococcus 
cultivated  from  the  pus  of  an  intra-abdominal  abscess. 

MICROCOCCUS  TETRAGENUS.-^LI  1881  Gaffsky42  discovered  a  micro- 
coccus  which  occurs  regularly  in  groups  of  four  or  tetrads.  He 


FIG.  45. — MICROCOCCUS  TETRAGENUS. 

first  isolated  it  from  the  pus  discharged  by  tuberculous  patients 
with  pulmonary  lesions.     Observed  in  smear  preparations  from  pus, 

40  Weichsellaum,  Baumgarten,  Jahresb.,  1899,  Ttef. 

**Veillon,  Compt.  rend.  soc.  de  biol.,  1893. 

42  Gaffsky,  Mitteil.  a.  d.  kais.  Gesundheitsamt,  i,  1881. 


STAPHYLOCOCCUS  PYOGENES  AUREUS          399 

the  tetrads  are  slightly  larger  in  size  than  the  ordinary  staphylo- 
coccus,  flattened  along  their  adjacent  surfaces,  and  surrounded  by 
a  thick  halo-like  capsule.  Preparations  from  cultures  often  lack 
these  capsules.  The  micrococcus  is  easily  stained  by  the  usual  basic 
aniliii  dyes.  Stained  by  Gram's  method,  it  is  not  decolorized,  retain- 
ing the  gentian-violet. 

Cultivation. — Micrococcus  tetrageiius  grows  on  the  ordinary 
laboratory  media,  showing  a  rather  more  delicate  growth  than  do 
the  staphylococci. 

On  agar,  the  colonies  are  at  first  transparent,  later  they  become 
grayish-white,  but  are  always  more  transparent  than  are  staphylo- 
coccus  cultures. 

On  gelatin,  growth  is  rather  slow  and  no  liquefaction  takes  place. 

Broth  is  evenly  clouded. 

On  potato  there  is  a  white,  moist  growth  which  shows  a  tendency 
to  confluence. 

Milk  is  coagulated  and  litmus  milk  indicates  acid  formation. 

Pathogenicity. — Micrococcus  tetragenus  is  especially  pathogenic 
for  Japanese  mice,  which  succumb  within  three  or  four  days  to  sub- 
cutaneous inoculation.43  Gray  mice,  rats,  guinea-pigs,  and  rabbits 
are  less  susceptible,  showing  only  a  localized  reaction  at  the  point 
of  inoculation.  The  organism  has  occasionally  been  isolated  from 
spontaneous  abscesses  observed  in  domestic  animals. 

In  man,  this  microorganism  is  usually  found  without  any  par- 
ticular pathogenic  significance,  in  sputum  or  saliva.  In  isolated 
cases,  however,  it  has  been  described  as  the  sole  incitant  of  abscesses. 

Bezanc.on44  has  isolated  Micrococcus  tetragenus  from  a  case  of 
meningitis.  A  single  case  of  tetragenus  septicemia  is  on  record, 
reported  in  1905  by  Forneaca.45 

In  America,  this  microorganism  has  not  been  frequently  observed 
in  connection  with  disease.  It  is  often  found,  however,  in  consider- 
able numbers,  in  smears  of  sputum  which  are  being  examined  for 
pneumococci  or  tubercle  bacilli. 

ENTEROCOCCUS. — This  is  a  capsulated  streptococcus  found  fre- 
quently in  the  intestines  of  infants,  and  first  described  by  Escherich 
and  Tavel.  It  is  a  pleomorphic  organism  which  may  appear  in  the 


"Miiller.  Wien.  klin.  Woch.,  17,  1904. 

44  Bezan^on,  Semaine  mod.,  1898. 

45  Forneaca,  Rif.  med.,  1903. 


400  PATHOGENIC   MICROORGANISMS 

stools  as  a  diplococcus  or  in  short  chains.  Even  in  the  diplococcus 
form,  the  individuals  may  be  of  different  size  and  shape,  some  of 
them  oval,  rather  than  round.  It  may  assume  diplobacillus-like 
forms. 

It  is  Gram-positive,  produces  neither  gas  nor  indol,  and  grows 
well  on  ordinary  broth  and  agar.  It  is  aerobic,  but  may  grow  under 
anaerobic  conditions. 

Its  relationship  to  intestinal  disease  in  children  has  been  sus- 
pected but  never  conclusively  proven.  Mice  are  susceptible  to  in- 
oculation, rabbits  and  guinea-pigs  less  so. 


CHAPTER   XXII 

THE  STREPTOCOCCI 

AMONG  the  pyogenic  cocci,  there  is  a  large  and  important  group 
of  organisms  which  multiply  by  division  in  one  plane  of  space  only, 
and  thus  give  rise  to  appearances  not  unlike  chains  or  strings  of 
beads.  The  term  streptococcus  or  chain-coccus  is,  therefore,  a  purely 
morphological  one  .which  includes  within  its  limits  microorganisms 
which  may  differ  from  each  other  considerably,  both  as  to  cultural 
and  pathogenic  properties.  Thus,  cocci  which  form  chains  may  be 
isolated  from  water,  milk,  dust,  and  the  feces  of  animals  and  man. 
These  may  have  little  but  their  morphological  appearance  in  common 
with  the  pyogenic  streptococci  which  are  so  important  as  the  incitants 
of  disease.  The  interrelationship  between  streptococci  from  different 
sources,  however,  is  by  no  means  fully  understood,  and  we  are  forced 
at  present  to  content  ourselves  with  the  recognition  of  a  large  mor- 
phological group,  in  no  individual  case  taking  the  pathogenic  or  more 
special  cultural  characteristics  for  granted. 

Of  paramount  importance  among  the  streptococci  are  those  which 
possess  the  power  of  giving  rise  to  disease  processes  in  animals  and 
in  man,  and  which,  because  of  their  frequent  association  with  sup- 
purative  inflammations,  are  roughly  grouped  under  the  heading  of 
Streptococcus  pyogenes. 

The  same  researches  upon  surgical  infections  which  led  to  the 
discovery  of  the  staphylococci  laid  the  basis  for  our  knowledge  of 
the  streptococci.  The  fundamental  studies  of  Pasteur  and  Koch1 
were  followed,  in  1881,  by  the  work  of  Ogston,2  who  was  the  first 
to  differentiate  between  the  irregularly  grouped  staphylococci  and 
the  chain-cocci. 

Pure  cultures  of  streptococci  were  first  obtained  by  Fehleisen3 
in  1883  and  by  Rosenbach4  in  1884.  The  thorough  and  systematic 


*Koch,  ' '  Untersuch.  iiber  Wundinfektion, "  etc.,  1878. 

2  Ogston,  Brit.  Med.  Jour.,  1881. 

'Fehleisen,  "Aetiol.  d.  Erysipelas,"  Berlin,  1883. 

*ltosenbach,   "Mikroorg.  bei  Wundinfektion,"  etc.,   Wiesbaden,   1884. 

401 


402 


PATHOGENIC   MICROORGANISMS 


researches  of  the  last-named  authors,  together  with  those  of  Passet,5 
were  of  special  influence  in  placing  our  knowledge  of  the  pathogenic 
properties  of  streptococci  upon  a  scientific  basis. 

Morphology  and  Staining. — The  individual  streptococcus  is  a 
spherical  microorganism  measuring  from  0.5  micron  to  1  micron  in 
diameter.  Since  the  line  of  cleavage  of  cocci,  when  in  chains,  is 


*• 


FIG.  46.  —  STREPTOCOCCUS  PYOGE'NES. 

perpendicular  to  the  long  axis  of  the  chain,  adjacent  cocci  often 
show  slight  flattening  of  the  contiguous  surfaces,  forming,  as  it 
were,  a  series  of  diplococci  arranged  end  to  end.  As  a  general  rule 
the  streptococci  pathogenic  for  man,  when  grown  upon  favorable 
media,  have  a  tendency  to  form  chains  made  up  of  at  least  eight 


6  Posset,  "Untersuch.  iiber  die  eitrigen  Phlegm./'  etc.,  Berlin,  1885. 


THE   STREPTOCOCCI  403 

or  more  individuals,  while  the  more  saprophytic,  less  pathogenic 
varieties  are  apt  to  he  united  in  shorter  groups.  Upon  this  basis  a 
rough  morphological  distinction  has  been  made  by  v.  Lingelsheim,6 
who  first  employed  the  terms  streptococcus  "longus"  and  "brcvis." 
A  differentiation  of  this  kind  can  hardly  be  relied  upon,  however, 
since  the  length  of  chains  is  to  some  degree  dependent  upon  cultural 
and  other  environmental  conditions.  Species  which  exhibit  long 
and  tortuous  chains,  when  grown  upon  suitably  alkalin  bouillon, 
or  ascitic  broth,  may  appear  in  short  groups  of  three  or  four,  or 
even  in  the  diplo  form,  when  cultivated  upon  solid  media  or  unfavor- 
able fluid  media. 

It  has  often  been  noticed  that  some  streptococci  will  form  cap- 
sules.7 We  are  not  referring  by  this  to  the  so-called  "streptococcus 
mucosus"  of  Schottmiiller,  which  is  now  spoken  of  as  "  pneumococcus 
mucosus"  or  Type  III,  but  ordinary  and  probably  true  hemolytic 
streptococci  may  on  occasion  form  capsules  which  are  noticeable  in 
smears  from  infected  animals  and  in  early  cultures  made  upon  media 
rich  in  animal  fluids,  but  may  be  lost  on  subsequent  transplantation 
to  simpler  media.  The  capsule  here  is  an  attribute  of  virulence. 
It  has  been  particularly  noticed  in  connection  with  the  so-called 
streptococcus  epidemicus  isolated  from  milk,  to  which  we  will  refer 
in  subsequent  paragraphs. 

Streptococci  do  not  form  spores,  are  non-motile,  and  do  not 
possess  flagella. 

An  astonishing  change  in  the  size  and  appearance  of  streptococci 
may  be  noticed  under  different  conditions  of  cultivation.  Strepto- 
cocci which  have  grown  under  anaerobic  or  partially  anaerobic  con- 
ditions will  often  show  chains  of  the  organisms  of  minute  size. 
Indeed,  we  have  seen,  interspersed  with  chains  of  the  ordinary  ap- 
p'earance,  individual  chains  composed  of  organisms  almost  as  small 
as  the  globoid  bodies  of  Noguchi.  It  is  these  small  individuals  which 
appear  under  anaerobic  conditions  that  have  been  responsible  for 
Rosenau's  ideas  concerning  the  etiological  role  of  streptococci  in 
poliomyelitis.  Again,  in  old  cultures  there  may  be  either  at  the 
ends  or  even  in  the  middle  of  the  chains,  large,  swollen  individuals, 
almost  as  big  as  small  yeast  cells.  When  these  are  first  encountered 

«v.  Lingelsheim,  "Aetiol.  u.  Therap.  d.  Streptok.  Infek."  Beit.  z.  Exp.  Ther., 
Hft.  1,  1899 

7  Pasquale,  Zieglers  Beit.,  xii;  Bordet,  Ann.  de  Plnst.  Pasteur,  1887;  Schott- 
nyller,  Munch,  med.  Woch.,  xx,  1903;  Hiss,  Jour.  Exp.  Med.,  vi,  1905. 


404  PATHOGENIC   MICROORGANISMS 

by  an  inexperienced  bacteriologist,  he  may  assume  for  some  time 
that  his  culture  has  been  contaminated.  These  swollen  individuals 
may  probably  be  interpreted  as  involution  forms. 

Streptococci  are  easily  stained  by  the  usual  anilin  dyes.  Stained 
by  the  method  of  Gram,  the  pyogenic  streptococci  are  not  decolorized 
and  invariably  retain  the  gentian-violet.  Certain  species  found  in 
stools  and  described  as  Gram-negative,  are  rare  and  are  non- 
pathogenic.  Others  of  the  "Streptococcus  brevis"  variety,  and 
purely  saprophytic,  may  stain  irregularly  by  the  Gram  method. 

Cultivation. — The  pyogenic  streptococci  are  easily  cultivated 
upon  all  the  richer  artificial  media.  While  meat  extract-pepton 
media  may  suffice  for  certain  strains,  it  is  usually  better  to  employ 
those  media  which  have  the  beef  or  veal  infusion  for  a  basis.  For 
the  cultivation  of  more  delicate  strains  of  streptococci,  especially 
when  taken  directly  from  the  animal  or  human  body,  it  is  well  to 
add  to  the  media  animal  albumin  in  the  form  of  whole  blood,  blood 
serum,  or  ascitic  or  pleural  transudates.  Glucose,  added  in  propor- 
tions of  one  to  two  per  cent,  likewise  renders  media  more  favorable 
for  streptococcus  cultivation.  Prolonged  cultivation  of  all  races  upon 
artificial  media  renders  them  less  fastidious  as  to  cultural  require- 
ments. The  most  favorable  reaction  of  media  for  streptococcus  cul- 
tivation is  moderate  alkalinity  (two-tenths  to  five-tenths  per  cent 
alkalinity  to  phenolphthalein) .  Growth  may  be  readily  obtained, 
however,  in  neutral  media  or  even  in  those  slightly  acid.  The 
optimum  temperature  for  growth  is  at  or  about  37.5°.  Above  43° 
to  45°  C.,  development  ceases.  At  from  15°  to  20°  C.,  growth,  while 
not  energetic,  still  takes  place,  an  important  point  in  the  differen- 
tiation of  these  microorganisms  from  pneumococci.  While  the  free 
access  of  oxygen  furnishes  the  most  suitable  environment  for  most 
races  of  streptococci,  complete  anaerobiosis  does  not  prevent  develop- 
ment in  favorable  media.  Strictly  anaerobic  streptococci  have  been 
cultivated  from  the  human  intestinal  tract  by  Perrone8  and  others. 

In  alkalin  bouillon  at  37.5°  C.,  pyogenic  streptococci  grow 
rapidly,  form  long  and  tortuous  chains,  and  have  a  tendency  to 
form  flakes  which  rapidly  sink  to  the  bottom.  Diffuse  clouding  occurs 
rarely  and  is  a  characteristic  rather  of  the  shorter  so-called  Strepto- 
coccus brevis.  When  sugar  has  been  added  to  the  broth  the  rapid 
formation  of  lactic  acid  soon  interferes  with  extensive  development. 

8  Perrone,  Ann.  de  Pinst.  Pasteur,  xix,  1905.  % 


THE  STREPTOCOCCI 


405 


This  may  be  obviated,  especially  when  mass  cultures  are  desired, 
without  sacrifice  of  the  growth-increasing  influence  of  the  glucose, 
by  adding  to  the  sugar-broth  one  per  cent  of  sterile  powdered  CaCO.9 

In  milk,  Streptococcus  pyo  genes  grows  readily  with  the  forma- 
tion of  acid,  followed,  in  most  cases,  by  coagulation  of  the  medium. 

On  agar-plates  at  37.5°  C.,  grow.th  appears  within  eighteen  to 
twenty-four  hours.  The  colonies  are  small,  grayish,  and  delicately 
opalescent.  They  are  round  with  smooth  or  very  slightly  corrugated 
or  lace-like  edges,  and  rise  from  the  surface  of  the  medium  in 
regular  arcs,  like  small  droplets  of  fluid.  Microscopically  they 
appear  finely  granular  and  occasionally,  under  high  magnification, 
may  be  seen  to  be  composed  of  long  in- 
tertwining loops  of  streptococcus  chains, 
which  form  the  lace-like  edges.  When 
ascitic  fluid  or  blood  serum  has  been 
added  to  agar,  growth  is  more  energetic 
and  the  colonies  correspondingly  more 
rapid  in  appearance  and  luxuriant  in 
development.  In  glucose-ascitic-agar, 
acid  formation  from  the  sugar  causes 
coagulation  of  albumin  with  the  conse- 
quent formation  of  flaky  white  precipi- 
tates throughout  the  medium.10 

In  gelatin  stab-cultures  growth  takes 
place  slowly,  appearing  after  twenty- 
four  to  thirty-six  hours  as  a  very 
thin  white  line,  or  as  disconnected 

little  spheres  along  the  line  of  the  stab.  The  colonies  on 
gelatin  plates  are  similar  in  form  to  those  on  agar,  but  are  usually 
more  opaque  and  more  distinctly  white.  The  gelatin  is  not  liquefied 
by  the  pyogenic  streptococci,  though  certain  of  the  more  saprophytic 
forms  may  occasionally  bring  about  slow  fluidification. 

On  Loeffler's  coagulated  blood  serum,  growth  is  rapid  and  luxuriant 
and  may  show  a  slight  tendency  to  confluence  if  the  medium  is  very 
moist.  Good  chain  formation  takes  place  on  this  medium. 

Upon  potatoes,  growth  is  said  not  to  take  place.11 

On  media  containing  red  blood  cells,  most  pathogenic  streptococci 


FIG.  47. — STREPTOCOCCUS  COL- 
ONY ON  SERUM  AGAR. 


9  Hiss,  Jour.  Exp.  Med.,  vi,  1905. 

10  Libman,  Medical  Record,  Ivii,  1900. 

11  Frosch  und  Kolle,  in  Fliigge,  ' '  Die  Mikroorganismen, ' '  1891. 


406  PATHOGENIC   MICROORGANISMS 

cause  hemolysis  and  decolorization.  It  is  useful  to  remember  this 
when  examining  blood-culture  plates,  for  here  the  yellow  transparent 
halo  of  hemolysis  and  decolorization  surrounding  the  colonies  may 
aid  in  differentiating  these  bacteria  from  pneumococci.  This  is  of 
especial  importance,  since  many  streptococci,  when  cultivated 
directly  out  of  the  human  blood,  do  not  exhibit  chain  formation, 
but  appear  as  diplococci. 

In  the  inulin-serum  media  of  Hiss,12  streptococci  do  not  produce 
acid  and  coagulation.  The  so-called  Streptococcus  mucosus,  a  cap- 
sule-bearing, inulin-fermenting  microorganism,  is  very  probably  a 
sub-species  of  the  pneumococcus  (see  later  section).  A  very  im- 
portant differential  characteristic  of  the  streptococci  as  a  class  is 
the  fact  that  they  are  not  bile  soluble.  This  distinguishes  them 
sharply  from  the  pneumococci.  The  test  is  carried  out  by  the 
method  of  Neufeld  described  in  the  chapter  on  pneumococci.  Ox 
bile  or  a  ten  per  cent  solution  of  taurocholate  of  sodium  is  added 
to  a  young  broth  culture  in  proportions  of  one  part  of  bile  or  tauro- 
cholate to  nine  parts  of  the  culture. 

Resistance. — Streptococci  on  the  ordinary  culture  media,  without 
transplantation  and  kept  at  room  temperature,  usually  die  out  within 
ten  days  or  two  weeks.  They  may  be  kept  alive  for  much  longer 
periods  by  the  use  of  the  calcium-carbonate-glucose  bouillon,  if  the 
cultures  are  thoroughly  shaken  and  the  powdered  marble  thoroughly 
mixed  with  the  bouillon  from  time  to  time.13  Preservation  at  low 
temperatures  (1°  to  2°  C.),  in  the  ice  chest,  considerably  prolongs 
the  life  of  cultures.  Virulence  is  preserved  longest  by  frequent 
transplantation  upon  albuminous  media.  In  sputum  or  animal 
excreta,  streptococci  may  remain  alive  for  several  weeks.  Strepto- 
cocci like  pneumococci  may  be  preserved  alive  and  with  virulence 
unchanged  by  drying  in  the  frozen  condition  by  the  method 
described  by  Swift,  and  referred  to  on  page  456.  For  ordinary 
purposes  preservation  in  tubes  of  defibrinated  rabbit's  blood  in  the 
ice-chest  is  the  most  practical  method. 

Streptococci  are  killed  by  exposure  to  a  temperature  of  54°  C. 
for  ten  minutes.14  Low  temperatures,  and  even  freezing,  do  not 
destroy  some  races. 


*- Hiss,  Jour.  Exp.  Me<lv  vi,  3005. 

13  Hiss,  loe.  cit. 

14  Stern'berg,  "Textbook  of  Bact.,"  2<1  e<L,  1901;  Hartmatni,  Arch.  f.  Hyg.,  vii. 


CLASSIFICATION   OF  STREPTOCOCCI  407 

The  action  of  various  chemical  disinfectants  lias  been  thoroughly 
investigated  by  v.  Lingelsheim/5  Avho  reports  among  others  the 
following  results:  Carbolic  acid  1:200  kills  streptococci  in  fifteen 
minutes.  In  the  same  time,  bichloride  of  mercury  is  efficient  in  a 
dilution  of  1 :1,500,  lysol  in  a  dilution  of  1 :200,  peroxide  of  hydrogen 
1 :35,  sulphuric  acid  1 :150,  and  hydrochloric  acid  1 :150.  Inhibition 
is  exerted  by  carbolic  acid  1 :550,  and  by  bichloride  of  mercury 
1 :65,000.  Exposure  to  direct  sunlight  kills  streptococci  in  a  few 
hours. 

CLASSIFICATION    OF    STREPTOCOCCI 

Since  the  first  discovery  of  the  chain  forming  cocci  there  has 
been  much  confusion  concerning  their  classification. 

The  Gram-positive  cocci  of  this  morphological  group  are  widely 
distributed  in  nature  and  vary  very  markedly  in  minor  cultural 
characteristics  and  virulence,  so  that  a  unification  of  the  group, 
as  it  has  been  possible  with  many  other  organisms,  has  been  prac- 
tically impossible.  The  earliest  observers  were  forced  to  abandon 
their  separation  of  the  streptococci  of  erysipelas  from  other  strepto- 
cocci because  of  the  work  of  Marbaix16  and  others,  who  produced 
erysipelas  in  rabbits  with  streptococci  from  non-erysipelatous  lesions, 
after  enhancement  of  their  virulence.  V.  Lingelsheim17  proposed  a 
purely  morphological  differentiation  of  "longus"  and  "brevis";  the 
former  class  including  the  streptococci  usually  found  in  pyogenic 
lesions  with  tendency  to  form  chains  of  six  or  more  links,  the  latter 
designating  the  short-chained  varieties,  including  the  less  virulent 
streptococci.  This  classification,  however,  is  not  tenable  because  of 
the  dependence  of  chain  formation  upon  reaction,  consistency,  and 
nutritive  qualities  of  the  media  employed  for  cultivation,  and  upon 
the  influence  of  animal  fluids  if  the  microorganisms  are  taken  direct 
from  lesions.  Schottmuller,18  in  1903,  proposed  a  classification  based 
both  upon  morphology  and  the  appearance  of  cultures  upon  human 
blood  agar.  By  this  method  he  divided  streptococci  into  two  main 

15  v.  Lingelsheim,  "Aetiol.  u.  Therap.  d.  Streptoe.  Inf.,"  etc.,  Beit.  z.  Exper. 
Therap.,  Hft.,  1,  1899. 
™Marbaix,  loc.  cit. 

17  v.   Lingelsheim,   "Aetiol.   u.  Therap.   d.   Streptokok.   Krankh.,"  etc.,   Berlin, 
1899. 

18  Schottmuller,  Munch,  med.  Woch.,  1903. 


408  PATHOGENIC   MICROORGANISMS 

groups  as  follows:  I.  Streptococcus  longus  or  haemolyticus,  consisting 
of  the  more  virulent  varieties,  with  tendency  to  form  long  chains,  and 
producing  hemolysis  upon  blood  media.  II.  Streptococcus  mitior  or 
viridans,  including  less  virulent  strains,  with  usually  shorter  chain- 
formation,  and  producing  green,  non-hemolyzing  colonies  upon  blood 
media.  A  third  group,  Streptococcus  mucosus,  will  receive  special  con- 
sideration in  a  separate  section,  and  is  probably  more  closely  related 
to  the  pneumococci. 

Another  nomenclature  has  recently  been  suggested  by  Smith  and 
Brown.19  These  workers  disapprove  of  the  use  of  the  name  "strep- 
tococcus viridans"  for  the  less  hemolytic  streptococci  because  many 
of  them  actually  produce  little  or  no  green  color  on  blood  agar, 
and  all  of  them  did  produce  more  or  less  hemolysis.  They  describe; 
two  types  of  streptococci  with  reference  to  hemolysis,  calling  the 
less  hemolytic  the  "Alpha"  type,  and  the  markedly  hemolytic  the 
"Beta"  type.  A  third  type,  "Gamma,"  produces  neither  discolora- 
tion nor  hemolysis.  They  describe  them  as  follows : 

Type  Alpha. — As  observed  after  forty-eight  hours  incubation  the  change 
produced  may  be  described  as  a  somewhat  greenish  discoloration  and  partial 
hemolysis  immediately  surrounding  the  colony,  forming  an  indefinitely 
bounded  zone,  1  to  2  millimeters  in  diameter,  and  surrounded  by  a  second 
narrow  clearer  and  not  discolorized  partially  hemolyzed  zone. 

Type  Beta. — Streptococci  of  this  type  produce  hemolyzed  zones  on  horse 
blood  agar  plates,  radically  different  from  those  of  the  Alpha  type.  They 
produce  clear  transparent,  completely  hemolyzed  colorless  zones,  2  to  4  milli- 
meters in  diameter  after  forty-eight  hours. 

Type  Gamma. — By  this  they  mean  streptococcus  strains  which  grow  within 
and  on  blood  agar  plates  without  the  production  of  perceptible  hemolysis 
or  discoloration.  Such  strains  are  those  described  by  Mandelbaum  as  strep- 
tococcus saprophyticus  and  correspond  to  some  of  the  Rosenow  types. 

Smith  and  Brown19  call  attention  to  the  great  care  which  is 
necessary  in  regard  to  details  of  cultivation  for  observing  strep- 
tococci for  their  hemolytic  properties  on  blood  plates.  They  con- 
tradict Ruediger's  assertion  that  the  formation  of  acid  in  such  plates 
may  give  rise  to  the  greenish  coloration.  There  is  no  essential  dif- 
ference in  result  between  plates  made  of  defibrinated  horse,  rabbit 

19  Smith  and  Brown,  Jour.  Med.  Res.,  31,  1914,  455,  and  Monograph  of  Rock 
Inst,,  No.  9,  January,  1919. 


CLASSIFICATION  OF  STREPTOCOCCI  409 

or  human  blood.  Meat  infusion  agar  is  more  favorable  than  meat 
extract  agar.  The  most  favorable  acidity  is  in  the  neighborhood  of 
1  per  cent  acid  to  phenolphthalein.  The  presence  of  dextrose  in 
the  medium  produces  a  marked  effect,  and  acid  hemolysis  may  result 
in  some  of  the  so-called  Gamma  types.  Very  small  amounts  of 
dextrose  in  blood  agar,  however,  may  lead  to  partial  or  complete 
inhibition  of  hemolysis  by  the  hemolytic  Beta  types.  The  reason 
that  the  Alpha  types  do  not  produce  green  zones  in  sugar-free  media 
is  explained  by  Smith  and  Brown  as  depending  upon  their  scant 
growth  in  such  media.  Under  anaerobic  conditions  Alpha  strepto- 
cocci and  pneumococci  simulate  Beta  type  zones. 

This  broad  classification  into  the  hemolyticus  ana  viridans  is  the 
practical  basis  on  which  bacteriologists  at  the  present  time  deal 
with  the  classification  of  pathogenic  streptococci.  Since,  however, 
bacteria,  morphologically  and  often  culturally  indistinguishable 
from  those  responsible  for  disease  processes,  can  be  found  present 
on  mucous  membranes  of  the  mouth,  intestines,  etc.,  in  many  normal 
persons  without  apparently  doing  the  slightest  harm,  a  great  deal 
of  effort  has  been  spent  in  attempts  to  discover  whether  cultural 
characteristics  could  in  any  way  be  related  to  pathogenic  properties. 
This  has  led  to  a  great  many  investigations  on  the  classification  of 
various  streptococci  by  carbohydrate  fermentation.  Such  studies 
have  been  made  by  Gordon,20  Houston,21  Andrewes  and  Horder,22 
Broadhurst,23  Winslow  and  Palmer,24  Hopkins  and  Lang,25  and 
Holman.26 

Gordon  found  ten  different  fermentation  reactions  among  twenty 
pyogenic  streptococci  examined,  and  forty-eight  different  fermenta- 
tion reactions  among  two  hundred  streptococci  isolated  from  saliva. 
Other  work  by  Andrewes  and  Horder  and  by  Buerger27  confirms  the 
irregularity  of  the  fermentation  reactions  within  this  group. 


20  Gordon,  Kep.  Med.  Off.  to  Local  Gov't  Bd.,  Great  Britain,  1902-3,  32  p.  421; 
ibid;   1903-4,  33,  p.  388. 

21  Houston,  Eep.  Med.  Off.  to  Local  Gov't  Bd.,  1903-4,  33,  p.  472. 
"Andrewes  and  Horder,  Lancet,  2,  1906,  708. 

23  Broadhurst,  Jour.  Infoc.  Dis.,  1912,  10,  272. 

24  Winslow  and  Palmer,  Jour.  Infec.  Dis.,  7,  1910,  1. 
**  Hopkins  and  Lang,  Jour.  Infec.  Dis.,   15,  1914,  63. 
26  Holman,  Jour.  Med.  Res.,  34,  1916,  377. 

"''Andrewes  and  Horder,  Lancet,  1906;  Buerger,  Jour.  Exp.  Med.,  ix,  1907. 


410  PATHOGENIC   MICROORGANISMS 

Andrewes  and  Horder  suggest  the  following  classification: 

(1)  Streptococcus  pyogenes.     Grows  in  long  chains  and  ferments  lactose, 
saccharose,  and  salicin;  does  not  coagulate  milk.     Streptococci  which  cause 
suppurative  lesions  or  severe  systemic  infections  belong  to  this  group. 

(2)  Streptococcus   mitis.     A   saprophytic  type  found  frequently  in  the 
mouth  which  shows  the   same   cultural  characteristics  as   the   streptococcus 
pyogenes,  but  grows  in  short  chains. 

(3)  Streptococcus  anginosus.    Found  frequently  in  throats  of  scarlet-fever 
patients  which  differs  from  the  pyogenes  only  in  coagulating  milk. 

(4)  Streptococcus  salivarius.    A  short -chain  type  which  ferments  lactose, 
saccharose,  and  raffinose,  and  coagulates  milk.     Streptococci  of  this  type  are 
found  frequently  in  the  mouth,  but  are  rarely  pathogenic. 

(5)  Streptococcus  fecalis.     A  short-chain  type  which   ferments   lactose, 
saccharose,  and  mannite.     This  type  is  found  normally  in  the  intestine,  and 
is  occasionally  pathogenic. 

(6)  Streptococcus  equinus.     A  short-chain  type  which  does  not  ferment 
lactose.     Found  in  horse  dung  and  never  pathogenic. 

Quantitative  determinations  of  the  amount  of  acid  formed  in 
various  sugars  by  different  races  have  also  been  made  by  Winslow 
and  Palmer28  and  others,  but  have  led  to  no  satisfactory  classi- 
fication. 

Studies  by  Hopkins  and  Lang  seem  to  show  that  the  streptococci 
found  in  most  human  infections  may  be  differentiated  from  the 
ordinary  saprophytic  types  by  the  fact  that  they  ferment  lactose 
and  salicin;  but  fail  to  ferment  raffinose,  inulin,  or  mannite.  Accord- 
ing to  their  results,  the  usual  saprophytic  types  found  in  the  mouth 
either  fail  to  ferment  salicin  or  ferment  raffinose  or  inulin,  whereas 
the  usual  fecal  types  ferment  mannite.  They  also  found  in  infection 
mannite  fermenters  which  were  apparently  of  fecal  origin.  Strep- 
tococci which  gave  the  same  fermentative  reaction  as  the  mouth 
saprophytes  were,  however,  frequently  found  in  malignant  endo- 
carditis. 

Holman  combined,  or  tried  to  combine  classification  by  the  Gor- 
don carbohydrate  fermentations  and  by  the  blood  agar  medium, 
and  somewhat  simplified  the  difficulties.  He  recognized  a  hemolytic 
and  a  non-hemolytic  group  under  each  of  which  he  classifies  eight 
fermentative  subgroups.  The  practical  importance  which  lie  at- 
taches to  his  investigations  is  thai  streptococci  from  similar  sources 


28  Jour,  of  Inf.  Dis.,  No.  viii,  1910,  1. 


CLASSIFICATION  OF   STREPTOCOCCI  411 

very  often  fall  into  identical  groups.  He  believes  that  by  his  method 
many  of  the  air  streptococci  could  be  traced  to  their  sources,  and 
that  similar  relation  between  source  and  cultural  characteristics 
could  be  found  for  streptococci  from  milk,  from  the  mouth,  from 
the  intestinal  tract,  and  from  animal  tissues.  He  concluded  that 
individual  groups  of  streptococci,  however,  were  not  specific  in  their 
disease  production. 

Cultural  classification  beyond  the  differentiation  into  hemolyticus 
and  viridans,  therefore,  has  not  helped  a  great  deal  in  characterizing 
the  streptococci  from  the  pathological  point  of  view.  It  has  been 
very  important,  therefore,  to  study  the  serological  relationships  of 
this  group,  a  problem  which,  apart  from  its  importance  in  pure 
classification,  has  fundamental  bearing  upon  the  questions  of  diagno- 
sis and  serum  therapy.  The  earlier  work  of  Aronson,29  Marmorek30 
and  others  showed  that,  although  the  serum  produced  with  any  race 
of  pyogenic  streptococci  frequently  exerted  considerable  agglutina- 
tive and  protective  action  against  other  strains,  these  relationships 
were  variable  and  that  antigenically  the  group  was  probably  poly- 
valent. There  was  much  confusion  at  first  concerning  these  matters, 
but  there  has  never  been  much  doubt  about  the  fact  that  sera  pro- 
duced with  single  strains  or  with  only  a  few  could  not  be  relied 
upon  to  react  with  any  chance  strain  obtained  from  a  case.  It  was 
not  until  quite  recently  that  more  light  has  been  shed  upon  this 
problem. 

In  order  to  deal  clearly  with  the  problem,  it  will  be  necessary  to 
consider  separately  the  viridans  group  and  the  hemolyticus  group. 
The  antigenic  relationship  between  the  two  groups  has  been  studied 
by  Kinsella31  who  finds  that  there  is  no  absolutely  sharp  antigenic 
line  between  viridans  and  hemolyticus  strains,  but  that  sera  immune 
to  hemolytic  streptococci  were,  in  a  definitely  limited  way,  related 
to  non-hemolytic  antigens.  On  the  other  hand,  sera  immune  to  non- 
hemolytic  strains  gave  no  positive  reactions  with  any  of  the 
hemolytic  antigens.  His  work,  however,  was  limited  in  the  number 
of  sera  used,  and  one  of  his  conclusions  was  that  the  hemolytic 
streptococci  are  probably  not  heterogeneous,  but  what  he  calls 
"unique."  Kinsella  and  Swift32  also  attempted  to  classify  non- 

29  Aronson,  Deut.  med.  Woch.,  29,  1903,  439. 

30  Marmorek,  Berl.  klin.  Woch.,  31,  1902,  299. 
n  Kinsella,  Jour.  Exper.  Med.,  28,  1918,  181. 

32  Kinsella  and  Swift,  Jour.  Exper.  Med.,  28,  1918,  169. 


412  PATHOGENIC   MICROORGANISMS 

hemolytic  streptococci  by  means  of  complement  fixation.  In  this 
work  they  used  a  number  of  strains  and  a  large  number  of  organisms, 
and  found  that  the  group  was  very  heterogeneous.  They  developed 
in  their  work  an  interesting  theory  as  to  the  inter-relationship  of 
these  heterologous  groups.  This  is  not  sufficiently  well  founded  to 
permit  our  going  into  it.  It  is,  however,  certain  from  their  work, 
and  from  that  of  others,  that  the  group  of  viridans  is  probably  as 
antigenically  heterologous  as  are  the  so-called  Type  IV  pneumococci. 
When  we  turn  to  the  hemolytic  group  we  find  that  a  more  hopeful 
state  of  affairs  seems  to  be  developing.  There  seemed  to  be  from 
the  beginning  a  certain  amount  of  immunological  grouping  in  organ- 
isms of  this  type.  Baginsky  and  Sommerfeld33  found  that  agglu- 
tinins  were  present  in  scarlet  fever  serum  which  seemed  to  be  specific 
for  other  strains  isolated  from  the  throats  of  scarlet  fever  patients, 
and  this  observation  was  confirmed  by  Moser  and  Von  Pirquet.34 
A  similar  apparent  similarity  between  strains  isolated  from  the 
blood  of  smallpox  cases  was  found  by  De  Waele  and  Sugg.35 
Neufeld36  suggested  that  there  was  a  relationship  between  agglu- 
tinability  and  virulence.  Kinsella  and  Swift37  studying  this  problem 
in  1918  came  to  the  conclusion  that  with  complement  fixation 
methods,  at  least,  the  hemolytic  variety  of  streptococcus  is  homo- 
geneous, consisting  of  members  that  are  almost  identical.  More 
recent  work,  however,  by  Dochez,  Avery  and  Lancefield38  seems  to 
have  shown  that  by  means  of  agglutination  it  may  be  possible  to 
classify  the  hemolytic  streptococci  into  immunological  sub-groups. 
They  define  four  biological  types  which  check  up  both  by  agglutina- 
tion reactions  and  by  protection  experiments.  They  encountered  at 
least  two  other  types,  and  indications  that  more  existed.  Their 
work  seems  well  founded  on  experimental  fact,  and  has  been  recently 
confirmed  to  this  extent  that  Bliss,  working  with  the  streptococci 
obtained  from  the  throats  of  scarlet  fever  cases  found  that  the 
organisms  from  such  sources  seemed  to  fall"  into  a  single  group. 
The  specificity  of  streptococci  has  also  been  recently  studied  by 


33  Baginsky  and  Sommerfeld,  Berl.  klin.  Woeh.,  37,  1900,  588. 
"Moser  and  Von  Pirquet,  Cent.  f.  Bakt.,  I,  1903,  34,  560. 

35  De  Waele  and  Sugg,  Arch.  Internal,  pharm.  et  therap.,  12,  1904,  205. 

36  Neufeld,  Zeit.  f .  Hyg.,  40,  1902,  54. 

37  Kinsella  and  Swift,  Jour.  Exper.  Med.,  28,  1918,  877. 

38  Doches,  Avery  and  Lancefield,  Jour.  Exper.  Med.,  30,  1919,  179. 


CLASSIFICATION   OF  STREPTOCOCCI  413 

Tunnicliff.39  She  has  given  particular  attention  to  absorption  tests, 
and  has  found,  in  agreement  with  Bliss,  that  the  hemolytic  strep- 
tococci from  scarlet  fever  form  a  distinct  group,  these  organisms 
removing  opsonins  and  agglutinins  for  these  cocci,  whereas,  ery- 
sipelas cocci  have  no  such  effect.  With  scarlet  fever  streptococci 
in  a  sheep,  Tunnicliff  produced  a  serum  which  agglutinated  hemoly- 
tic streptococci  in  dilutions  of  1  to  2000,  and  opsonized  them  in 
dilutions  of  1  to  360.  She  found  that  this  serum  protected  mice 
against  the  hemolytic  streptococci  isolated  from  scarlet  fever  pa- 
tients, but  not  from  erysipelas,  mastoiditis  and  influenza.  This  pro- 
tective powder  was  rapidly  lost  by  the  sheep  serum,  but  was  restored 
by  the  addition  of  normal  sheep  serum.  Her  further  work  indicates 
that,  like  the  scarlet  fever  cocci,  the  erysipelas  cocci  form  a  distinct 
group,  also. 

As  we  shall  see,  the  difficulty  with  these  investigations  has  largely 
been  technical  in  that  the  spontaneous  agglutination  of  the  hemolytic 
streptococci  makes  it  very  difficult  to  carry  out  reliable  agglutination 
work.  These  difficulties  will  be  alluded  to  in  connection  with  ag- 
glutination. 

Virulence  and  Pathogenicity. — Different  races  of  pyo  genie  strep- 
tococci show  considerable  variations  in  virulence,  and  there  are  few 
organisms,  pathogenic  both  for  animals  and  man,  which  show  com- 
parable fluctuations  in  virulence.  The  character  or  severity  of  the 
lesion  in  man  gives  little  evidence  as  to  the  virulence  of  the  organism 
for  animals.  Such  differences  are,  to  a  certain  extent,  dependent 
upon  inherent  individual  characteristics,  but  are  rather  more  likely 
to  be  the  consequences  of  previous  environment  or  habitat.  Pro- 
longed cultivation  upon  artificial  media  usually  results  in  the  reduc- 
tion of  the  virulence  of  a  streptococcus,  while  an  originally  low  or 
reduced  virulence  may  often  be  much  enhanced  by  repeated  passage 
of  the  streptococci  through  animals.  It  is  noteworthy,  however, 
that  while  the  passage  of  a  streptococcus  through  rabbits  will  usually 
enhance  its  virulence  for  susceptible  animals  in  general,  repeated 
passages  through  mice  may  increase  the  virulence  for  these  animals 
only,  even  occasionally  depressing  the  virulence  for  rabbits. 

Among  the  domestic  animals,  those  most  susceptible  to  experi- 
mental streptococcus  infection  are  white  mice  and  rabbits.  Guinea- 
pigs  and  rats  are  less  easily  infected,  and  the  larger  domestic  animals, 

30  Tunnicliff,  Jour,  of  the  A.  M.  A.,  75,  1920,  1339. 


414  PATHOGENIC   MICROORGANISMS 

cattle,  horses,  goats,  cats,  and  dogs,  are  relatively  refractory.  Al- 
most complete  immunity  toward  streptococcus  infections  prevails 
among  birds. 

The  nature  of  the  lesions  following  experimental  animal  inocula- 
tion depends  upon  the  manner  of  inoculation,  the  size  of  the  dose 
given,  and  most  of  all  upon  the  grade  of  virulence  of  the  inoculated 
germ.  Subcutaneous  inoculations,  according  to  the  virulence  of  the 
inoculated  material,  may  result  in  a  simple  localized  abscess,  differ- 
ing from  a  staphylococcus  abscess  only  in  the  more  serous  nature 
of  the  exudate  and  the  frequent  occurrence  of  edema,  or  in  a  severe 
general  septicemia  with  a  hardly  noticeable  local  lesion.  Subcu- 
taneous inoculation  of  mice  may  result  in  general  sepsis  followed 
by  death  within  thirty-six  to  forty-eight  hours,  or  less,  with,  the 
presence  of  streptococci  in  the  heart's  blood  and  the  viscera.  Intra- 
pleural  or  intraperitoneal  inoculation  of  susceptible  animals  with 
virulent  streptococci  leads  usually  to  a  peculiarly  hemorrhagic  form 
of  exudate,  due  both  to  the  diapedesis  caused  by  the  violent  inflam- 
matory process,  and  to  the  hemolysis  of  the  red  cells  by  the  strepto- 
coccic  hemolysins.  Inoculation  of  rabbits  at  the  base  of  the  ear 
with  virulent  streptococci  may  result  in  the  formation  of  a  lesion 
indistinguishable  histologically  from  erysipelas  in  man.40  Marbaix41 
has  shown  that  such  erysipeloid  lesions  could  be  produced  in  rabbits 
by  streptococci  from  various  and  indifferent  sources,  provided  that 
the  virulence  of  each  strain  could  be  sufficiently  enhanced. 

Intravenous  inoculation  of  rabbits  with  virulent  cultures  usually 
results  in  a  rapidly  fatal  septicemia.  An  animal  which  has  died  of 
a  streptococcus  infection  usually  shows  serosanguineous  edema  about 
the  point  of  inoculation,  multiple  hemorrhagic  spots  upon,  the  serous 
membranes,  and  congestion  of  the  viscera.  The  microorganisms  can 
almost  invariably  be  found  in  the  heart's  blood,  in  the  spleen,  and 
in  the  exudate  about  the  inoculated  area.  Microscopically,  when 
the  process  has  lasted  sufficiently  long,  parenchymatous  degeneration 
of  all  the  organs  may  be  observed.  In -the  more  chronic  infections 
articular  and  periarticular  lesions  may  occur.42 

Spontaneous  streptococcus  disease  seems  to  occur  among  some 
of  the  larger  domestic  animals. 


Fehlciscn,  loc.  cit.;  Franlccl,  Cent.  f.  Bakt.,  vi. 
Marbaix,  La  Cellule,  1892. 

,  Zeit.  f.  Hyg.,  ui;  Hiss,  Jour,  Mod,  Res.,  xix,  1908. 


CLASSIFICATION   OF  STREPTOCOCCI  415 

Thus,  in  horses,  mules  and  donkeys,  there  is  an  acute  contagious 
disease  of  the  upper  air  passages,  colloquially  known  as  "strangles" 
(in  German,  Druse)  due  to  the  streptococcus  equinus,  a  iioii-hemolys- 
ing,  salicin  fermenting  strain.  The  etiological  significance  of  this 
organism  was  first  recognized  by  Schlitz1;5  and  lias  been  subsequently 
confirmed.  The  disease  attacks  chiefly  young  animals,  is  charac- 
terized by  fever  and  general  systemic  depression,  with  subsequently 
an  acute  catarrh  of  the  nasal  and  pharyngeal  mucosa,  with  local 
glandular  swelling.  Often  the  submaxillary  gland  may  be  involved, 
and  pneumonia  may  ensue.  In  cattle,  streptococci  are  most  fre- 
quently associated  with  the  inflammations  of  the  udders.  Strepto- 
coccus mastitis  may  seriously  effect  the  quality  and  quantity  of  the 
milk,  and  perhaps  has  very  definite  relationship  to  disease  in  human 
beings,  a  matter  to  which  we  will  refer  in  a  subsequent  paragraph. 
Occasionally,  post-partum  uterine  inflammation  in  cows  may  be  caused 
by  streptococci. 

In  chickens  there  occurs  a  form  of  septicemia  caused  by  a 
streptococcus  which  is  described  by  Moore44  as  being  an  anaerobe 
which  does  not  liquefy  gelatine,  does  not  coagulate  milk,  forms 
acid  in  alkalin  broth  and  grows  in  flaky  masses  like  a  streptococcus 
hemolyticus  in  broth  cultures.  Moore  does  not  state  whether  it 
produces  hemolysis  or  not.  The  disease  has  a  sudden  onset,  and  is 
usually  fatal.  It  is  apparently  transmitted  from  chicken  to  chicken. 

Among  the  smaller  laboratory  animals,  occasional  streptococcus 
infections  may  be  observed  in  rabbits.  Recently  an  epidemic  disease 
among  white  mice  due  to  streptococcus  was  studied  by  Kutscher.45 
As  a  rule,  however,  streptococcus  disease  is  by  far  more  rare  among 
animals  than  it  is  among  human  beings. 

STREPTOCOCCUS  INFECTIONS  IN  MAN. — In  man,  a  large  variety  of 
pathological  processes  may  be  caused  by  streptococci  and  here 
again  the  nature  of  the  infection,  whether  definitely  localized  or 
generally  distributed,  depends  upon  the  relationship  existing  be- 
tween the  virulence  of  the  incitant  and  the  resistance  of  the  subject. 

Superficial  cutaneous  infections  are  frequently  caused  hy  strep- 
tococci and  these  in  the  milder  cases  may  be  similar  to  the  localized 
abscesses  caused  by  staphylococci.  In  severe  cases,  however,  infec- 


43  Schutz,  Arch.  f.  Thier,  Heilk.,  14,  1889,  172. 

44  Moore,  Pathology,  etc.,  of  Infec.  Diseases  in  Animals,  MacMillan,  New  York, 
1916. 

45  Kutscher,  Cent.  f.  Bakt.,  xlvi. 


416  PATHOGENIC   MICROORGANISMS 

tion  is  followed  by  rapidly  spreading  edema,  lymphangitis,  and 
severe  systemic  manifestations  with  the  development  of  a  grave 
cellulitis,  often  threatening  life  and  requiring  energetic  surgical 
interference. 

The  particular  significance  of  streptococci  in  surgically  infected 
wounds  and  the  effect  of  their  presence  upon  therapeutic  procedures 
is  considered  in  another  section  dealing  with  the  bacteriology  of 
infected  wounds. 

Suppurations  of  bone  may  be  caused  by  streptococci,  and  con- 
stitute a  severe  form  of  osteomyelitis.  Such  lesions  when  occurring 
in  the  mastoid  bone  are  not  infrequently  secondary  to  streptococcus 
otitis  and  may  lead  to  a  form  of  meningitis  which  is  in  most  cases 
fatal. 

Streptococcus  meningitis  fortunately  does  not  occur  very  often, 
but  when  it  does  occur  is  usually  fatal.  Most  cases  are  probably 
secondary  to  such  lesions  as  otitis  and  mastoiditis  mentioned  above, 
but  occasionally  primary  streptococcus  meningitis  may  occur  in  the 
course  of  bronchopneumonia.  We  have  seen  a  number  of  cases 
in  which  streptococci  were  associated  in  the  spinal  fluid  with  in- 
fluenza bacilli. 

As  mentioned  above,  erysipelas  is  a  streptococcus  hemolyticus 
disease.  It  was  first  isolated  from  such  lesions  by  Fehleisen46  who 
believed  that  the  organism  obtained  by  him  was  a  particular  variety, 
specific  in  erysipelas.  He  named  it,  for  this  reason,  streptococcus 
erysipelatis.  Subsequently,  however,  it  was  shown  by  Marbaix47 
and  Petruschky48  and  others  that  erysipelas-like  lesions  could  be 
produced  in  animals  with  streptococci  from  many  other  sources. 
The  production  of  the  peculiar  erysipelas  lesion  seems  to  depend, 
on  the  one  hand  upon  the  relative  virulence  of  the  strain  and  upon 
the  infection  of  the  lymphatics  of  the  skin.  The  streptococci  in  this 
disease  are,  according  to  MacCallum,49  located  in  the  crevices  of 
the  tissues  and  the  lymph  channels  of  the  skin.  A  peculiarity  is 
that,  unlike  streptococcus  lesions  in  other  places,  in  this  disease  the 
inflammatory  exudate  consists  very  largely  by  mononuclear  cells. 


48  Fehleisen,  Aeriol.   d.  Erysipelas,  Berlin,  1883. 
47  Marbaix,  La  Cellule,  1892. 

™PetrusMy,  Zeit.  f.  Hyg.,  13. 

49  MacCallum,  Textbook  of  Pathology,  Second  Edition,  1920  •  Monograph  of  the 
Eock.  Inst.,  No.  10,  1919. 


CLASSIFICATION  OF  STREPTOCOCCI  417 

This  disease  is  particularly  dangerous  in  infants  in  whom  it  may 
spread  over  the  entire  surface  of  the  body. 

It  was  formerly  supposed  that  a  number  of  different  forms  of 
acute  enteritis  might  be  due  to  streptococci,  but  this  has  never  been 
positively  demonstrated,  and  is  doubtful.  However,  streptococcus 
infections  of  the  walls  of  the  intestines  by  passage  into  the  sub- 
mucosa  may  occasionally  occur. 

The  inflammation  which  is  known  as  Ludwig's  angina  is  usually 
of  a  streptococcus  hemolyticus  nature.  In  this  condition  the  origin 
is  usually  from  a  focus  in  the  teeth,  tonsils  or  pharynx,  or  perhaps 
a  peritonsular  abscess,  and  consists  of  a  violent,  acute  inflammation 
of  the  areolar  tissues  of  the  submaxillary  region  of  the  neck. 

Superficially,  infections  through  the  skin,  through  abrasions  and 
injuries  are  among  the  most  frequent  and  dangerous  infections 
caused  by  the  hemolytic  streptococcus.  These  are  particularly  fre- 
quent among  surgeons  and  pathologists,  and  their  course  and  out- 
come is  determined  entirely  by  the  relationship  between  virulence 
of  the  strain  and  resistance  of  the  individual.  Given  a  sufficiently 
virulent  strain,  a  very  minute  abrasion  or  injury  with  the  inocula- 
tion of  a  small  quantity  of  the  organisms  only,  may  suffice  to  result 
in  a  rapid  and  fatal  infection.  We  know  of  one  autopsy  infection 
in  which  a  minute  pin  prick  through  a  rubber  glove,  hardly  visible 
to  the  eye,  resulted,  within  twelve  hours,  in  high  fever,  and  within 
twenty-four  hours,  in  delirium  with  little  red  lymphatic  lines  run- 
ning up  the  arm  from  the  source  of  the  lesion.  In  such  cases,  the 
local  lesion  may  look  relatively  innocent,  since,  with  sufficient 
virulence,  the  point  of  inoculation  may  show  nothing  more  than  a 
small  red  swelling  which  is  soggy  and  edematous  and  looks  quite 
different  from  the  slower  processes,  with  central  abscess  formation, 
caused  by  the  less  virulent  streptococci  and  staphylococci. 

Puerperal  sepsis  is  one  of  the  most  dangerous  of  the  infections 
caused  by  the  hernolytic  streptococci,  but  with  the  advent  of  more 
perfect  obstetrical  methods,  this  grave  infection  of  the  uterus  is 
becoming  more  and  more  rare. 

From  all  of  the  streptococcus  lesions  a  general  infection  of  the 
blood  stream  may  arise,  if  the  infection  proceeds  with  sufficient 
virulence.  Although  both  the  viridans  and  the  hemolyticus  strains 
may  be  responsible  for  such  septicemic  conditions,  the  most  fre- 
quent and  dangerous  ones  are  those  caused  by  the  hemolyticus. 
We  will  devote  a  separate  paragraph  to  a  description  of  the  viridans 


418  PATHOGENIC   MICROORGANISMS 

septicemia  accompanying  endocarditis.  Hemolyticus  septicemia, 
secondary  to  wound  infection,  puerperal  septicemia  or  other  lesions, 
are  usually  initiated  by  grave  symptoms  of  fever,  chill  and  general 
depression,  and  the  organisms  can  be  isolated  in  blood  cultures 
taken  in  agar  plates  or  in  hormone  broth.  It  is  an  important 
thing  to  remember  that  in  all  of  these  septicemias  the  presence  of 
the  organisms  in  the  blood  may  not  signify  that  they  are  actually 
multiplying  in  the  blood.  It  is,  in  our  opinion,  more  likely  that 
the  organisms  at  first  simply  enter  the  blood  stream  from  the  lesion, 
and  are  destroyed  in  the  circulation.  If,  during  this  period,  the 
focus  can  be  surgically  cleaned  out,  rapid  recovery  may  follow. 
We  have  seen  cases,  in  which  the  surgical  lesion  was  accessible  to 
recover  promptly  after  operation,  although  blood  cultures  taken 
before  had  shown  numerous  organisms  in  the  blood  stream.  In 
cases  dead  of  streptocococcus  septicemia,  the  organisms  may  be  cul- 
tivated from  all  the  organs  and  from  the  heart's  blood. 

In  dealing  with  other  streptococcus  lesions  in  man,  it  will  be 
convenient  for  us  to  differentiate  sharply  between  those  caused  by 
the  hemolyticus  and  those  due  to  the  viridans  strains.  Since  normal 
human  beings  harbor  hemolytic  streptococci  in  the  mouth  and 
pharynx  in  a  large  percentage  of  individuals  examined,  it  is  natural 
that  inflammations  of  the  upper  respiratory  tract  should  frequently 
be  due  to  streptococci.  Thus,  the  hemolytic  streptococci  are  fre- 
quently the  causative  agents  in  pharyngitis,  and  are  often  associated 
with  the  more  severe  forms  of  follicular  tonsilitis.  Some  of  these 
tonsillar  infections  may  be  accompanied  by  high  fever,  and  severe 
illness,  and  the  local  inflammation  may  be  so  severe  that  it  cannot 
be  clinically  differentiated,  with  certainty,  from  diphtheria.  From 
such  throat  infections,  septicemia  does  not  often  follow,  but  occa- 
sionally severe  generalized  infections  may  ensue.  MacCallum 
describes  the  case  of  a  physician,  one  of  our  colleagues,  who  had 
repeated  attacks  of  throat  infection  caused  by  streptococci.  In  one 
of  these  he  developed  glands  of  the  throat  which  were  incised  and 
streptococcus  pus  was  found.  A  year  later  there  was  a  sudden 
recurrence  with  a  rapidly  developing  general  septicemia,  and  a 
scarlatiniform  rash.  He  died  within  a  few  days.  In  connection 
with  this,  it  is  interesting  to  note  that  we  have  seen  three -or  four 
cases  which  occurred  among  soldiers  in  France  who,  in  the  course 
of  severe  streptococcus  throat  infections  developed  rashes  Hint  were 
difficult  to  distinguish  from  those  of  scarlet  fever.  Just  what  the 


CLASSIFICATION   OF   STREPTOCOCCI  419 

explanation  of  these  eruptions  is,  we  cannot  say.  They  occur  in 
fatal  septicemias  of  many  varieties,  but  are  particularly  associated 
with  fatal  streptococcus  infection. 

In  scarlet  fever,  hemolytic  streptococci  are  invariably  found 
associated  with  and  probably  the  causative  agents  of  the  severe 
angina  that  regularly  accompanies  this  disease.  This  was  observed 
by  Loeffler  in  1884,  and  has  been  confirmed,  since  then,  by  many 
observers.  The  regularity  of  these  findings  has  been  such  that  it 
has  led  to  the  assumption  that  the  hemolytic  streptococci  might  be 
etiologically  related  to  scarlet  fever  itself.  This  is  a  problem  which 
has  been  much  discussed,  but  cannot  be  thoroughly  gone  into  with- 
out an  extensive  review  of  the  literature.  We  may  say  that  the 
general  weight  of  evidence  is  at  the  present  time  distinctly  against 
such  an  assumption,  and  that  it  is  most  likely  that  the  streptococci 
in  scarlet  fever  are  the  most  regular  and  severe  secondary  invaders. 
Yet,  it  must  be  admitted  that  no  conclusive  denial  of  the  possibility 
can  be  made  on  the  basis  of  experimental  evidence.  The  hemolytic 
streptococci  not  infrequently  invade  the  blood  stream  of  scarlet 
fever  patients  in  the  course  of  the  disease.  Baginsky  and  Sommer- 
feld50  found  them  in  the  blood  at  autopsy,  and  Anthony51  and  others 
have  confirmed  this^  Anthony  found  them  in  the  heart's  blood  of 
ten  out  of  eighteen  autopsies.  An  important  fact  first  noted  by 
Moser  and  Von  Pirquet52  is  the  apparent  antigenic  identity  of  strains 
of  hemolytic  streptococci  isolated  from  different  cases  of  scarlet 
fever.  Bliss  has  recently  confirmed  this  by  the  newer  and  more 
reliable  methods  of  agglutination  developed  by  Dochez  and  Avery.53 

In  diphtheria  and  in  smallpox  the  hemolytic  streptococci  are 
frequently  found  as  secondary  invaders.  Gay,54  who  has  carefully 
analyzed  the  literature  on  streptococcus  infection,  cites  a  number 
of  observers  who  have  found  the  organisms  in  the  blood  streams 
of  fatal  cases  of  smallpox,  and  in  some  of  those  that  recovered. 

It  Jias  long  been  known  that  the  dangerous  bronchopneumonias 
which  occur  in  the  course  of  measles  and  influenza  may  be  of  strep-, 
tococcus  origin.  Eyre53  has  made  particular  studies  of  this  condition 


and  Sommcrfeld,  Borl.  klin.  Woeli.,  27,   ]900. 
ii,  Jour.  Infec.  Dis.,  6,  1909,  332. 
K Moser  and    I'on  Pirquet,  Zeit,  f.  Bakt.,  34,  1903,  500  and  714. 
**  Dochcs  and  Aver;/,  and  Lmn-c/icltl,  Journ.  Exp.  Mod.,  vol.  30,   1919,  p.  179. 
Cl  (!<iii.  Jour,  of  Lab.  and  Clin.  Mod.  3,  1918,  721. 
•"""'  K I/re,  Jour,  of   Bnctor.   and   Pathol.,   14,   1910,   160. 


420  PATHOGENIC   MICROORGANISMS 

in  measles  bronchopiieumonias,  and  for  both  measles  and  influenza 
much  information  has  been  gathered  during  the  late  war.  It  appears 
that  both  measles  and  influenza,  as  well  as  perhaps  some  other  mild 
infections  of  the  upper  respiratory  tract,  render  the  individual 
tremendously  susceptible  to  secondary  infection.  Under  conditions 
such  as  those  developed  in  the  camps  during  the  war,  these  strep- 
tococcus bronchopneumonias  may  become  epidemic,  probably  by 
reason  of  the  generalized  interchange  of  mouth  organisms  among 
susceptible  individuals  crowded  together  under  camp  conditions. 
Epidemiologically,  it  is  of  interest  to  note  that  under  these  condi- 
tions the  carrier  rate  of  hemolytic  streptococci  reaches  high  per- 
centages. Irons  and  Marine,56  at  Camp  Ouster,  found  70  per  cent 
of  the  individuals  examined  to  be  carriers  of  these  organisms,  and 
Levy  and  Alexander57  in  certain  regiments  found  89  per  cent  to  be 
carriers.  Given,  at  the  same  time,  extensive  outbreaks  of  measles 
and  influenza,  the  conditions  for  widespread  secondary  streptococcus 
pneumonias  are  established.  It  is  not  impossible  that  widespread 
ward  infection  may  occur,  in  that  patients  who  enter  hospitals  with 
measles  and  influenza,  even  without  being  carriers  of  hemolytic 
streptococci,  may  pick  them  up  in  the  hospitals  from  adjacent  beds, 
from  doctors  or  from  nurses,  possibilities  which  indicate  the  great 
importance  of  prompt  removal  of  cases  developing  pneumonias  from 
measles  and  influenza  wards,  the  careful  hygiene  of  the  mouths 
and  throats  of  such  cases,  isolation  of  beds  by  screening,  and  the 
wearing  of  masks  by  doctors  and  nurses,  not  so  much  for  their  own 
protection  as  for  that  of  the  susceptible  patient.  MacCallum,  Cole 
and  Dochez58  made  careful  studies  of  the  streptococcus  pneumonias 
occurring  at  some  of  the  camps.  The  pathological  facts  resulting 
from  this  study  are  reported  by  MacCallum  in  the  10th  Monograph 
of  the  Rockefeller  Institute,  issued  in  1919.  According  to  him,  the 
streptococci  seem  to  extend  downward  into  the  smaller  bronchioles, 
giving  rise  to  intensive  inflammations  in  the  air  passages,  then  ex- 
tending into  the  network  of  lymphatics  surrounding  the  bronchioles 
and  the  pleura.  There  was  a  rapid  production  of  pleurisy  and 
empyema,  with  hemorrhage  about  the  bronchioles  and  a  very  curious 
infiltration  of  the  alveolar  walls  themselves  with  leucocytes  chiefly 


66  Irons  and  Marine,  Jour.  A.  M.  A.,  70,  1918,  687. 

57  Levy  and  Alexander,  Jour.  A.  M.  A.,  1918,  1827,  Vol.  70. 

68  MacCallum,  Cole  and  Dochez,  Jour.  A.  M.  A.,  70,  1918,  1146. 


THE  STREPTOCOCCI  421 

of  the  mono-nuclear  variety.  The  appearance  of  these  processes, 
according  to  MacCallum,  is  quite  different  from  that  seen  in  the 
ordinary  forms  of  bronchopneumonia. 


EPIDEMIC  SORE  THROAT  DUE  TO  MILK  INFECTION 

Sore  throat  epidemics  traceable  to  milk  have  been  observed  in 
England  since  1875.  The  onset  of  these  cases  is  usually  accompanied 
by  sudden  chilliness,  with  muscular  soreness,  headache  and  nausea. 
The  cases  are  strikingly  similar  to  the  milder  forms  of  influenza. 
The  first  carefully  observed  epidemic  in  this  country  occurred  in 
Boston  in  1911,  and  was  epidemiologically  studied  by  Winslow.59 
There  were  48  fatal  cases  in  the  Boston  epidemic.  Since  that  time 
a  number  of  similar  epidemics  have  been  described,  one  of  the  most 
extensive  being  that  which  took  place  in  Chicago  in  1911  and  was 
studied  by  Capps  and  Miller,60  and  by  Davis  and  Rosenow.61  In 
the  Chicago  epidemic  there  were  10,000  cases,  hardly  any  of  which 
came  from  the  west  side  of  the  city.  The  relationship  to  the  milk 
supply  was  carefully  studied.  Of  622  cases  investigated,  87  per  cent 
or  537  used  milk  from  a  certain  dairy,  and  79  per  cent  of  the  fatal 
cases  used  the  same  milk.  People  taking  milk  from  this  dairy  were 
fourteen  times  more  numerous  than  those  getting  it  from  other 
sources.  Of  153  nurses  in  a  certain  hospital  using  the  milk,  80  per 
cent  got  the  disease,  while  of  721  in  other  hospitals,  only  4.8  per  cent 
came  down.  There  was  a  coincident  epidemic  of  sore  throats  among 
the  employees  of  the  dairy  where  bovine  mastitis  was  found  in  the 
cows.  In  fact,  almost  5  per  cent  of  the  cows  of  this  dairy  had 
mastitis,  and  streptococci  were  isolated  from  the  milk  of  a  cow 
and  from  the  throat  of  a  girl  on  the  same  farm.  Davis  and  Rosenow 
describe  the  organisms  isolated  from  these  cases.  In  all  of  them 
they  found  a  streptococcus  which  produced  large  colonies  on  blood 
agar,  larger  than  the  ordinary  hemolytic  organisms.  There  was 
moderate  hemolysis,  and  the  organism  was  virulent  for  guinea  pigs, 
mice  and  rabbits.  Capsules  were  developed  on  animal  passage. 
They  believed  their  organism,  which  they  called  streptococcus  epi- 
demicus,  to  be  a  distinct  species.  However,  Davis,  in  a  subsequent 

59  Winslow,  Jour.  Infec.  Dis.,  10,  1912,  73. 

80  Capps  and  Miller,  Jour.  A.  M.  A.,  58,  1912,  1848. 

61  Davis  and  Eosenow,  Jour.  A.  M.  A.,  58,  1912,  773. 


422  PATHOGENIC   MICROORGANISMS 

paper62  declares  the  organism  to  be  indistinguishable  from  other 
hemolytic  streptococci. 

VIRIDANS  INFECTIONS. — The  non-hemolytic  or  so-called  green, 
streptococci  are,  as  stated  above,  more  likely  to  be  associated  with 
the  less  severe  and  subacute  lesions.  These  organisms  are  usually 
harmless  saprophytes. 

They  are  associated  frequently  with  tooth  abscesses,  may  be 
present  in  middle  ear  disease  and  infections  of  the  accessory  sinuses 
of  the  nose.  They  are  not  infrequently  found  in  the  tonsillar  crypts 
and  have  been  known  to  cause  mild  forms  of  tonsillitis. 

The  viridans  is  frequently  associated  with  subacute  vegetative 
endocarditis.  In  such  cases  the  organisms  cause  relatively  firm 
vegetations,  usually  on  the  mitral,  but  sometimes  also  on  the  aortic 
valves,  extending  occasionally  along  the  walls  of  the  auricle.  The 
organisms  can  be  readily  cultivated  from  the  blood  stream  where 
they  may  remain  for  weeks  and  months.  Often,  in  such  cases  the 
resistance  of  the  patient  is  unusually  high  for  a  long  time,  the 
bacteria  probably  being  disposed  of  in  the  blood  stream,  but  are 
passed  either  constantly  or  intermittently  into  the  blood  stream 
from  the  infected  valves.  Death  may  ensue  after  weeks  or  months. 
Death  usually  follows  by  a  gradual  wearing  down  of  the  patient's 
resistance.  Cerebral  or  renal  embolism  may  occur,  and  joint  con- 
ditions may  develop  secondarily. 

It  has  been  suggested  that  the  frequent  association  of  rheumatism 
and  cardiac  lesions  may  be  due  to  the  fact  that  the  green  streptococci 
are  responsible  for  both  conditions.  An  enormous  amount  of  rather 
confusing  work  has  been  done  upon  the  etiology  of  rheumatism 
from  this  point  of  view.  It  is  a  fact  that  many  coccus  infections, 
and,  for  that  matter,  other  forms  of  general  infection,  may  lead 
to  secondary  joint  lesions  in  both  animals  and  man.  Poynton  and 
Paine63  in  1900  described  a  diplococcus  not  very  dissimilar  from 
viridans  strains  which  they  obtained  from  eight  cases  of  acute  rheu- 
matic fever,  and  with  which  they  claimed  that  they  could  produce 
lesions  in  rabbits  which  they  considered  typical  of  rheumatism. 
The  organism  was  recovered  from  the  blood  and  pericardial  fluids 
of  their  patients.  They  describe  them  as  minute  diplococci,  grown 
best  in  acid  media  under  anaerobic  conditions,  but  capable  of  growth 


62  Davis,  Amer.  Jour.  Pub.  Health,  8,  40,  cited  after  Gay,  loc.  cit. 

63  Poynton  and  Paine,  Lancet,  London,  1900,  860  and  932. 


THE  STREPTOCOCCI  423 

on  the  surface  of  ordinary  media.  Recently,  Rosenow64  reported 
the  isolation  of  streptococcus  viridans  from  the  joints  of  seven  cases 
of  articular  rheumatism.  He  also  claimed  that  he  could  produce 
non-suppurative  arthritis,  endocarditis  and  pericarditis  in  rabbits 
with  these  cultures.  He  described  the  organisms  as  intermediate 
in  character  between  the  ordinary  viridans  and  the  hemolyticus. 
These  claims  have  led  to  a  great  deal  of  subsequent  experimentation 
and  speculation.  There  is  no  question  about  the  fact  that  viridans 
strains  have  often  been  isolated  from  cases  of  rheumatism,  but  it 
is  not  unlikely  that  equal  care  in  similar  attempts  would  have 
succeeded  in  isolating  these  ubiquitous  organisms  from  many  other 
groups  of  disease.  Swift  and  Kinsella65  have  made  a  particularly 
careful  study  of  this  problem,  and,  though  working  with  great  care 
and  experience,  obtained  the  viridans  from  only  8.3  per  cent  of 
58  cases.  In  no  case  did  they  obtain  the  organisms  from  the  joints 
themselves,  and  we  ourselves  can  attest  to  the  fact  that  the  large 
majority  of  direct  cultures  taken  from  rheumatic  joints  prove  quite 
sterile.  While,  therefore,  the  role  of  the  viridans  in  endocarditis 
is  clear,  its  relationship  to  rheumatic  fever  is  entirely  unproven  at 
the  present  writing. 

The  same  uncertainty  exists  concerning  its  association  with  an- 
other disease  which  is  frequently  classified  with  the  two  diseases 
mentioned,  namely,  chorea.  A  number  of  writers,  notably  Quigley,66 
have  found  the  viridans  in  the  blood  of  chorea  cases,  but  nothing 
more  definite  than  this  has  been  noted. 

Rosenow,67  too,  has  recently  claimed  that  there  was  an  associa- 
tion between  viridans-like  streptococci  and  poliomyelitis.  In  the 
special  section  on  poliomyelitis  in  this  book  we  will  deal  with  the 
globoid  or  coccoid  bodies  isolated  by  Flexner  and  Noguchi.68  Some 
two  or  three  years,  after  these  observations  were  made,  Rosenow 
and  Towne,69  as  well  as  Mathers,70  reported  that  they  had  found 
streptococci  of  the  viridans  group  in  the  central  nervous  system  of 
cases  of  poliomyelitis.  Rosenow  and  Wheeler71  grew  these  cocci 


"Rosenow,  Jour.  A.  M.  A.,  60,  1123  and  61,  1947  and  2007. 
65  Swift  and  Kinsella,  Arch.  Inter.  Med.,  19,   1917,  381. 
"Quigley,  Jour.  Infec.  Dis.,  22,  1918,  198. 

67  Rosenow,  'Jour.  Infcc.  Dis.,  22,  1918,  379. 

68  Flexner  and  Noguchi,  Jour.  Expcr.  Med.,  18,  1913,  461. 
e"  A'o.srnow  and  Townc,  Jour.  Med.  Research,  36,  1917,    175. 
711  Mather*,  Jour.  A.  M.  A.,  '67,  1916,  1019. 

71  Eosenow  and  Wheeler,  Jour.  Infec.  Dis.,  22,  1918,  281, 


424  PATHOGENIC   MICROORGANISMS 

under  anaerobic  conditions,  and  found  that  minute  forms  developed, 
small  enough  to  be  filtered,  and  they  believed  that  it  was  these 
anaerobic  minute  forms  that  represented  the  coccoid  bodies  of 
Noguchi.  It  is  very  difficult  to  give  any  conclusive  judgment  on 
this  matter.  Bull  could  not  confirm  Rosenow's  work,  and  showed 
that  the  organisms  isolated  by  him  were  neither  uniform  in  cultural 
behavior,  nor  had  they  any  specificity  as  regards  the  production 
of  lesions  in  animals.  The  weakest  parts  of  Rosenow's  work  are 
his  animal  experiments  in  which  he  produced  lesions  in  guinea  pigs, 
dogs  and  monkeys  with  these  cultures;  none  of  these  animals  have 
been  found  susceptible  to  poliomyelitis  virus  derived  directly  from 
the  filtered  brain  tissues  of  human  cases.  Mistakes  are  quite  likely 
to  occur  since  cocci  similar  to  the  Rosenow  types  may  be  found 
in  a  considerable  number  of  rabbits,  and  even  in  monkeys  dead  of 
a  variety  of  conditions,  unless  cultures  are  taken  before  or  very  soon 
after  death. 

Personally  we  do  not  believe  that  Rosenow  is  right. 

The  Question  of  Tissue  Specificity  of  the  Viridans. — Rosenow,  in 
the  course  of  the  work  cited  above,  isolated  viridans  streptococci 
not  only  from  poliomyelitis  brains  and  rheumatic  cases,  but  also 
from  gastric  ulcers,  and  in  the  course  of  this  work  he  developed 
the  theory  that  individual  strains  of  these  organisms  may  acquire 
a  specific  affinity  for  particular  tissues.  His  idea  is  that  organisms 
isolated  from  rheumatic  lesions,  gastric  lesions,  etc.,  will  selectively 
lodge  in  analogous  tissues  on  animal  injection  or  on  gaining  entrance 
to  another  individual.  Other  observers  have  not  been  able  to  confirm 
this  claim,  and,  indeed,  it  is  very  unlikely  that  such  a  thing  occurs. 
At  any  rate,  no  definite  proof  has  been  brought,  and  the  weight 
of  evidence  is  against  his  claim.  It  is  an  interesting  thought,  yet 
a  dangerous  one  to  spread  broadcast,  since  it  has  influenced  clinical 
thinking  to  an  extent  not  warranted  by  experimental  fact.  In  the 
specific  localization  of  organisms  in  the  tissues,  it  seems  to  us  much 
more  likely  that  tissue  factors  are  paramount,  such  as  perhaps 
specific  hypersusceptibility,  or  local  reduction  of  resistance.  Such 
a  thought  is  indicated  by  the  work  of  Faber72  who  sensitized  joints 
with  extracts  of  streptococci,  subsequently  producing  lesions  in  these 
joints  by  intravenous  injection  of  the  microorganisms  themselves. 
None  of  these  ideas,  however,  have  been  proven,  and  the  entire 
question  remains  one  of  the  difficult,  unsettled  problems. 

72  Faber,  Jour.  Exper.  Med.,  22,  1915,  615. 


THE   STREPTOCOCCI  425 

Toxic  Products. — In  spite  of  extensive  researches  by  many  in- 
vestigators upon  the  nature  of  the  poisons  produced  by  streptococci, 
our  understanding  of  these  substances  is  still  very  incomplete.  The 
grave  systemic  symptoms  so  often  accompanying  comparatively 
slight  streptococcus  lesions  argue  strongly  for  the  production  by 
these  microorganisms  of  a  powerful  diffusible  poison.  Toxic  nitrates 
of  streptococcus  cultures  have  indeed  been  obtained  by  Roger,73 
Marmier,74  Baginsky  and  Sommerfeld,75  Marmorek,76  and  many 
others;  but  these  have  in  no  case  been  comparable  in  potency  to 
the  soluble  toxins  of  diphtheria  or  of  tetanus.  When  injected  into 
young  guinea-pigs  in  sufficient  quantity,  these  nitrates  produce  rapid 
collapse  and  death. 

Aronson77  in  1902  found  that  nitrates  from  streptococcus  cultures 
made  in  various  ways  were  practically  without  toxicity,  although 
he  occasionally  obtained  toxic  symptoms  by  giving  very  large  doses 
of  two  to  four-day  cultures.  The  residue  of  streptococcus  body 
substances  was  not  toxic  at  all.  Simons78  in  1904  found  a  similar 
lack  of  toxicity  of  the  bodies  of  the  organisms,  but  claimed  that 
nitrates  of  broth  cultures  two  to  nine  days  old  produced  emaciation 
and  diarrhea  in  rabbits.  He  claimed  that  it  was  necessary  to  grow 
the  organisms  in  the  presence  of  animal  exudates  or  leucocytes,  a 
claim  which  corresponds  to  Marmorek79  ideas.  Braun80  found  some 
toxicity  of  nitrates  of  streptococcus  cultures  for  rabbits.  More 
recently,  Clark  and  Fenton81  cultivated  hemolytic  streptococci  upon 
Locke's  solution  containing  defibrinated  blood  and  0.5  per  cent 
glucose.  They  claimed  that  0.5  to  1  c.c.  of  the  filtrates  of  such 
cultures  killed  rabbits  with  considerable  regularity. 

We  ourselves  have  made  similar  studies  of  streptococcus  poisons 
and  were  unable  to  confirm  the  claim  of  Clark  and  Fenton  though 
following  the  very  same  method.  We  have  found,  however, 
that  cultures  of  various  hemolytic  streptococci  made  upon  hormone 
broth,  and  similar  broth  with  defibrinated  blood,  grown  for  from 

73  Eager,  Kev.  de  med.,  1892. 

74  Marmier,  Ann.  de  1'inst.  Pasteur,  ix,  1895,  p.  533. 
'"'Baginsky  und  Sommerfeld,  Berl.  klin.  Woch.,  ]900. 
T6  Marmorek,  Berl.  klin.  Woch.,  1902. 

"Aronson,  Berl.  klin.  Woch.,  1902,  39. 

78  Simons,  Cent.  f.  Bakt.,  35,  1904. 

79  M armor ek,  Ann.  de  PInst.  Past.,  16,  1902,  169. 

80  Braun,  Cent.  f.  Bakt.,  61,  1912,  383. 

81  Clark  and  Fenton,  Jour.  A.  M.  A.,  71,  1918,  1048. 


426  PATHOGENIC   MICROORGANISMS 

eighteen  to  twenty-four  hours  under  partially  anaerobic  conditions, 
contained  definite  toxic  substances.  Filtered  through  Berkefeld 
candles  and  injected  into  rabbits  in  quantities  ranging  from  3  to  6 
cubic  centimeters,  sickness  consisting  of  general  muscular  weakness, 
respiratory  difficulty,  and  in  about  8  per  cent  of  the  cases  death 
ensued.  Similar  toxic  substances  could  be  obtained  by  repeatedly 
washing  young  agar  growths  of  streptococci  in  salt  solution  and 
filtering  immediately.  We  do  not  regard  these  as  specific  exotoxins, 
but  believe  them  to  be  entirely  similar  to  substances  obtained  in 
the  same  way  from  young  influenza  cultures,  typhoid,  dysentery, 
meningococcus  and  prodigiosus  cultures.  The  work  on  these  sub- 
stances is  not  yet  completed.  They  are  not  antigenic  and  not  specific, 
and  repeated  injection  into  rabbits  in  sublethal  amounts  produces 
chronic  emaciation  and  usually  death  after  five  or  six  injections. 
They  may  well  have  important  bearing  on  the  symptoms  of  strepto- 
coccus diseases.  They  are  certainly  neither  specific  exotoxins  nor 
endotoxins,  and,  for  the  present,  we  speak  of  them,  for  lack  of  a 
better  term,  as  "X"  substances.82 

STREPTOCOCCUS  HEMOLYSIN. — The  production  of  hemolysin  by 
streptococci  has  been  mentioned  briefly  in  connection  with  our  sec- 
tion on  classification,  since  this  property  has  become  one  of  the 
important  criteria  of  differentiation.  The  substance  responsible  for 
the  laking  of  red  blood  corpuscles,  being  a  secretion  of  the  organ- 
isms, comparable  to  the  true  toxins  of  some  other  bacteria,  has  been 
made  the  subject  of  a  considerable  number  of  investigations.  The 
observation  of  this  phenomenon  was  first  made  by  Marmorek83  in 
1895.  Marmorek  believed  that  there  was  a  direct  relation  between 
virulence  and  hemolytic  power.  Other  investigators,  however, 
notably  Schottmuller,84  believed,  from  the  beginning,  that  the 
hemolytic  power  was  a  constant  characteristic  of  certain  strains, 
unchangeable  by  experimental  enhancement  or  reduction  of  the 
virulence.  As  mentioned  before,  the  streptococcus  hemolysins  may 
be  conveniently  observed  by  cultivation  of  the  organisms  on  blood 
agar  plates.  The  criteria  for  media,  blood  and  reaction  have  been 
mentioned  above.  The  hemolysins  may  also  be  obtained  in  liquid 
media  by  the  filtration  of  young  liquid  cultures.  Apparently  it  is 

82  Zinsser,   Jour,   of   Immunol.,   5,    1920,   No.    3,   p.   265 ;    Zinsser,  ParTcer   and 
Kuttner,  Transac.  Soc.  Exp.  Med.  and  Biol.,  Dec.,   1921. 
*3Marmorelc,  Ann  de  Plnst.  Past.,  1895. 
84  Schottmilllcr,  Mun.  med.  Woch.,  1903. 


THE   STREPTOCOCCI  427 

advantageous  that  media  for  this  purpose  should  contain  ascitic 
fluid  or  serum.  The  first  to  obtain  streptolysin  in  this  was 
Besredka.85  The  nature  of  the  filter  used  for  this  purpose  makes 
a  considerable  amount  of  difference,  and  it  is  stated  by  De  Kruif 
and  Ireland80  that  the  Maassen  filters  are  the  most  suitable  ones, 
and  that  even  with  these  considerable  amounts  of  hemolysin  are  lost 
in  filtration.  According  to  Besredka,  Braun87  and  others,  the  strep- 
tolysin is  not  a  very  stable  substance,  and  for  this  reason  it  is 
necessary  to  filter  the  cultures  at  a  point  of  optimum  growth.  The 
same  observation  has  been  made  by  M'Leod88  who  obtained  the  best 
results  by  filtering  serum  broth  cultures  after  sixteen  to  eighteen 
hours'  growth.  A  careful  experimental  study  of  the  substance  has 
recently  been  made  by  DeKruif  and  Ireland  in  the  paper  cited 
above.  They  paid  particular  attention  to  the  correlation  of  strep- 
tolysin production  to  the  logarithmic  growth  curve  of  the  organisms. 
The  broth  used  by  them  in  most  of  their  experiments  consisted  of  a 
2  per  cent  pepton  beef  infusion  broth,  brought  to  a  of  7.8  withp^. 
sodium  carbonate.  To  this  broth,  following  the  methods  of  M'Leod 
and  others,  animal  serum  in  various  concentration  was  added.  The 
best  yields  were,  obtained  with  sheep  serum  at  a  concentration  of 
20  per  cent,  although  similar  concentrations  of  human  and  rabbit 
serum  gave  good  yields. 

By  avoiding  filtration  and  using  the  high-power  centrifuge,  the 
ordinary  loss  of  hemolysin  incident  to  filter  adsorption  can  be 
avoided.  The  hemolytlc  effects  can  then  be  titrated  against  various 
types  of  red  corpuscles  by  adding  the  supernatant  fluid  or  filtrate 
in  graded  quantities  to  0.5  c.c.  of  a  2.5  per  cent  suspension  of  washed 
cells. 

De  Kruif  and  Ireland's  results,  in  the  main,  confirm  the  observa- 
tions of  others,  namely,  that  the  hemolysin  appears  early  in  the 
cultures,  and  when  the  broth  flasks  are  seeded  with  young  agar 
cultures  the  peak  may  be  reached  at  from  seven  to  eight  hours. 
When  the  crest  of  the  growth  curve  has  been  passed,  the  lysin 
begins  to  decrease  and  may  disappear  completely  in  fourteen  hours. 

According  to  Braun,  the  streptolysin  is  extremely  sensitive  to 
heat.  Six  hours  exposure  at  37°  seemed  to  destroy  it.  Six  hours 


a,  Ann.  de  1'Tnst.Past.,  15,  1901,  880. 
M  Di'Kmif  and  Ireland,  Jour.  In  foe.  Bis.,  20,  1920,  285. 
*  Rr<niii,  Out.  f.  P.akt.,  62,  1912,  383. 
"*  M'Leod,  Jour.  Pathol.  and  Baeter.,  16,  1912,  321. 


428  PATHOGENIC   MICROORGANISMS 

at  room  temperature  caused  decided  reduction,  but,  kept  in  the 
ice-box  for  this  period,  reduction  was  slight  only. 

The  substance  is  not  apparently  specific  for  any  one  variety  of 
cell,  but  as  produced  from  most  strains  it  may  effect  the  red  cells 
of  human  beings,  of  mice  and  rabbits  of  sheep  and  of  horses,  indis- 
criminately, though  perhaps  with  quantitative  differences.  Both 
Besredka  and  Braun  failed  to  produce  an  anti-lysin  for  the  strepto- 
coccus substance.  The  nature  of  the  anti-lytic  properties  possessed 
by  some  normal  sera  is  not  entirely  clear.  Since  it  is  an  extremely 
heat  stable  substance,  being  but  incompletely  destroyed  at  100°  C., 
it  has  been  suggested  that  it  could  not  be  an  antibody  in  the  ordinary 
sense  of  the  work,  but  may  consist  of  lypoidal  substances  like  choles- 
trin  in  the  blood  stream. 

STREPTOLEUCOCIDIN. — In  1905  Ruediger89  observed  that  filtrates 
of  virulent  streptococci  would  prevent  the  phagocytosis  of  non- 
virulent  streptococci  by  leucocytes.  The  same  thing  was  affirmed 
by  Hektoen.90  Recently,  Nakayama91  has  made  more  detailed 
studies  of  streptoleucocidin.  He  finds  that,  as  a  general  rule, 
hemolytic  and  non-hemolytic  streptococci,  when  non-virulent,  and 
easily  taken  up  by  phagocytosis,  under  the  influence  of  normal  serum, 
do  not  produce  leucocidin.  Virulent  strains,  however,  which  are  not 
phagocytable  produce  leucocidin  in  demonstrable  quantities.  In 
cultures  grown  on  10  per  cent  serum  broth,  the  largest  quantity 
of  leucocidin  was  produced  in  from  ten  to  twenty-four  hours,  after 
which  production  fell  off.  The  largest  quantities  were  obtained  in 
broth  containing  goat  serum  and  horse  serum.  There  was  a  definite 
relationship  between  the  volume  of  production  and  virulence.  The 
substance  was  rendered  inactive  by  heating  at  58°  to  60°  for  thirty 
minutes.  It  was  unstable  on  preservation,  and  could  not  be  reac- 
tivated by  the  addition  of  small  quantities  of  fresh  culture  fluid. 
Normal  serum  and  leucocytic  extract  possess  some  anti-leucocydal 
power,  a  property  which  is  destroyed  by  heating  at  70°  for  thirty 
minutes. 

He  mentions  that  immunization  with  streptococci  did  not  produce 
anti-leucocydal  properties,  but  injections  with  the  leucocydal  culture 
fluids  were  successful.  The  streptoleucocidin  seemed  to  be  distinct 


89  Euediger,  Jour.  A.  M.  A.,  44,  1905,  198. 
<JO  Hektoen,  Jour.  A.  M.  A.,  46,  1906,  1407. 
91  Nakayama,  Jour.  Infec.  Dis.,  27,  1920,  86. 


THE   STREPTOCOCCI  420 

from  the  streptolysin,  and  the  antibody  against  it  did  not  neutralize 
streptoleucocidin. 

Nakayama  used  the  following  method  for  the  determination  of  leucocidin, 
a  method  which  is  given  because  it  can  be  applied  to  tests  for  similar 
substances  from  many  organisms.  His  procedure  was  as  follows :  Leucocytes 
were  obtained  by  injecting-  aleuronat  as  usual.  When  rabbits  were  used  the 
aleuronat  was  injected  into  the  pleura.  After  twelve  hours,  the  animals 
were  bled,  and  the  exudate  withdrawn  after  opening  the  chest,  and  mixed 
with  equal  amounts  of  1.5  per  cent  sodium  citrate  solution  to  prevent  coag- 
gulation. 

A  solution  of  methylene  blue  is  made  as  follows:  1  c.c.  methylene  blue, 
20  c.c.  absolute  alcohol,  and  29  c.c.  distilled  water. 

Various  leucocytic  suspensions  are  made  up  with  0.9  per  cent  salt  solution, 
and  the  mixtures  made  up  to  2  c.c.  There  are  progressively  fewer  leucocytes 
in  the  successive  mixture.  Two  drops  of  methylene  blue  solution  are  then 
added  to  each  tube  and  the  mixture  covered  with  a  layer  of  liquid  paraffin. 
The  tube  is  then  put  in  the  incubator  at  37°  for  two  hours.  If  reduction 
occurs,  the  solution  becomes  colorless ;  if  no  reduction  occurs,  the  color  remains 
green.  This  would  make  the  minimum  quantity  of  leucocytes  which  cause 
a  reduction  of  the  methylene  blue.  Then,  to  test  the  leucocidin  different 
quantities  of  the  leucocydal  fluid  are  added  to  twice  the  minimum  quantity 
of  leucocytic  suspension  which  has  caused  reduction  in  the  above  test.  These 
mixtures  are  then  incubated  for  one  and  one-half  hours  at  37°,  and  at  the 
end  of  this  period,  two  drops  of  methylene  blue  are  added  and  the  tubes 
again  covered  with  liquid  paraffin.  Again  they  are  incubated  for  two  hours 
and  readings  are  made.  If  the  tube  contains  a  green  color,  it  indicates  that 
something  has  prevented  reduction. 

Antibodes  and  Immunization. — For  reasons  not  wholly  under- 
stood at  present,  recovery  from  streptococcus  infection  does  not  to 
any  marked  degree  produce  immunity  against  these  bacteria.  Active 
immunity  may,  however,  be  produced  in  rabbits,  goats,  horses,  and 
other  domestic  animals  by  treatment  with  gradually  increasing  doses 
of  streptococcus  cultures.92 

In  carrying  out  such  immunizations  it  is  necessary  to  use  for  the 
first  injection  attenuated  or  dead  bacteria.  Attenuation  may  be 
accomplished  by  moderate  heating  or  by  the  addition  of  chemicals 
(terchloride  of  iodin).  Neufeld93  advises,  for  the  first  injection  in 
immunizing  rabbits,  the  use  of  ascitic-broth  cultures  killed  by  heat- 

92  Koch  und  Petruschky,  Zeit.  f .  Hyg.,  xxiii,  1896. 
™  Neufeld,  Zeit,  f.  Hyg.,  xliv,  1903. 


430  PATHOGENIC    MICROORGANISMS 

ing  to  70°  C.  This  is  followed,  after  ten  days,  by  a  second  injection 
of  a  small  quantity  of  fully  virulent  cocci.  Following  this,  injections 
are  made  at  intervals  of  ten  days  with  constantly  increasing  doses. 
Modifications  of  these  general  principles  arc  employed  in  most 
laboratories. 

The  sera  of  animals  so  treated  contain  no  demonstrable  antitoxic 
or  antihemolytic  substances.94  It  has  been  claimed  that  these  sera 
exerted  demonstrable  bactericidal  power  both  in  vitro  and  in  vivo. 
Personally,  we  doubt  the  validity  of  this  claim  considerably,  since 
bactericidal  action  upon  Gram-positive  cocci  by  body  fluids  unaided 
by  leucocytes,  is  not  likely  to  occur.  In  all  such  experiments  we 
believe  errors  have  been  made,  due  to  the  agglutination  of  organisms 
and  the  subsequent  diminution  of  colonies  when  the  tests  were  made 
by  plating  methods.  The  opsonic  powers  of  such  sera,  however, 
cannot  be  doubted,  and  enhancement  of  phagocytosis  has  been 
demonstrated,  both  in  vitro  and  in  vivo.  In  fact,  it  was  with  such 
sera  that  Denys  made  his  important  observations  on  the  importance 
of  serum  in  phagocytosis,  showing  that  the  immune  bodies  in  the 
serum  acted  upon  the  bacteria  and  not  upon  the  leucocytes. 

The  protective  value  of  streptococcus  immune  sera  for  infected 
animals  is  considerable,  reaching  often  a  potency  hardly  explicable 
by  the  demonstrable  bactericidal  or  opsonic  power,  and  thereby 
suggesting  some  other  active  factor  not  understood  as  yet.95  Aron- 
son96  has  produced  immune  sera  by  the  treatment  of  horses  with  a 
streptococcus  derived  from  a  case  of  scarlatina,  0.0004  c.c.  of  which 
sufficed  to  protect  mice  from  ten  times  the  fatal  dose  of  a  strepto- 
coccus culture.  These  high  protective  values,  however,  are  obtained 
only  when  the  serum  injections  are  given  simultaneously  with  the 
bacteria.  Given  four  or  six  hours  after  infection,  much  higher 
dosage  must  be  employed  and  protective  results  are  much  less 
regular  in  occurrence.97  Other  antistreptococcic  sera  have  been 
produced  by  Denys,  Menger,  Tavel,  and  others,  all  showing  more 
or  less  marked  potency  in  protecting  animals.98 

94  Lingelsheim,  Zeit.  f.  Hyg.,  x,  1891. 

95  Denys  et  Marchand,  "Mecanisme  de  I'immunite, "  etc.,  Brussels,  1896. 

96  Aronson,  Berl.  klin.  Woch.,  xxxii,  1896;  ibid.,  xlii  and  xliii,  1902;  ibid.,  viii 
and  ix,  1905. 

97  Denys,  "Le   Serum  antistreptoc., "  Louvain,   1896;    Van  de    I'elde,  Ann.  de 
Tlnst.  Pasteur,  1896. 

w  Denys  et  Marchand,  Bull,  de  1'acad.  roy.  de  med.  de  Belgique,  :J898;  Menger, 
Berl.  klin.  Woch.,  1902;  Tavel,  Corr.-Bl.  f.  Schw.  Aertze. 


THE   STREPTOCOCCI  431 

Since  these  sera,  while  in  a  general  way  potent  against  all  strep- 
tococci, have  been  found  protective  chiefly  against  the  specific  micro- 
organism employed  for  their  production,  Van  de  Velde,"  Denys, 
Aronsoii,  and  others  had  advised  the  immunization  of  the  animal 
with  a  large  variety  of  streptococcus  races,  derived  from  many 
different  human  sources.  The  resulting  "polyvalent"  serum  is  more 
apt  to  exert  equally  high  protective  powers  against  all  streptococcus 
infections.  The  therapeutic  value  of  such  sera  in  the  treatment  of 
human  infections  is  still  sub  judice.  Undeniably  favorable  reports 
are  published  each  year  in  increasing  number,  but  are  by  no  means 
regular  or  comparable  to  the  results  obtained  in  diphtheria  with 
diphtheria  antitoxin.  Nevertheless,  in  mild  cases  or  in  those  in  which 
the  lesions  have  been  distinctly  localized,  the  sera  seemed  to  be  suffi- 
ciently useful  to  justify  their  use  and  necessitate  their  standardization. 
The  recent  antigenic  classification  of  streptococci  by  Dochez  and  Avery 
may  considerably  alter  our  procedures  in  regard  to  the  polyvalence  of 
serum.  It  may  become  possible  in  the  future  to  type  the  infecting 
streptococci  and  to  use  a  corresponding  serum. 

Standardization  of  sera  in  regard  to  their  protective  power  is 
accomplished  by  the  methods  first  devised  by  Marx100  for  the 
standardization  of  swine-plague  serum,  and  depends  upon  the  ability 
of  the  serum  to  protect  animals  against  a  measured  dose  of  virulent 
streptococci.  Aronson101  designates  as  a  "normal  serum"  one  of 
which  0.01  c.c.  will  protect  a  mouse  against  ten  to  one  hundred 
times  the  fatal  dose  of  virulent  streptococci.  One  c.c.  of  this  serum 
equals  one  serum  unit.  Comparisons  by  animal  experiment  with  this 
standard  serum  approximately  determine  the  value  of  other  sera. 

In  titrating  the  protective  power  of  sera,  it  is  of  course  necessary 
to  remember,  as  we  have  stated  above,  that  streptococci  fall  into 
antigenic  groups  and  a  serum  produced  with  one  strain  or  a  mixture 
of  strains  may  occasionally  have  no  action  whatever  upon  a  given 
strain  from  a  case.  The  methods  of  testing  protective  power  on 
mice  are  becoming  so  important  in  all  kinds  of  bacteriological 
research,  that  we  think  it  important  to  insert  some  of  the  technical 
details  as  described  by  Dochez,  Avery  and  Lancefield  in  the  paper 


"  \-(in  de  Veldc,  Arch,  dc  mod.  exper.,  1897. 
'""  Marx,  Deutsche  thierarzt.  Woch.,  vi,  1901. 

wl  Aronson,  Borl.  klin.  Woch.,  xliii,  1902;   Otto,  Arb.  a.  d.  konigl.   Just.,  etc., 
Frankfurter  a  M.,  Heft  2,  1906. 


432 


PATHOGENIC    MICROORGANISMS 


quoted  above.102  .In  order  to  do  protection  experiments  properly, 
it  is  necessary,  in  the  first  place,  to  produce  powerful  serum;  in 
the  second  place,  it  is  important  to  raise  the  virulence  of  the  test 
strain  to  a  high  point.  We  may  state  from  our  own  experience 
that  not  all  streptococci  can  be  raised  to  the  same  degree  of  potency 
for  mice.  Dochez  and  Avery  raised  the  virulence  of  a  number  of 
strains,  by  continuous  mouse  passage,  to  a  point  at  which  0.000001 
to  0.00000001  c.c.  of  a  broth  culture  would  kill  the  animals  in  twenty- 
four  to  forty-eight  hours.  Workers  in  our  own  laboratory  have 
occasionally  been  able  to  do  this  with  individual  strains,  but  most 
strains  could  not  be  raised  to  a  virulence  approaching  this.  The 
organisms  are  best  grown  in  ascitic  broth.  0.5  c.c.  of  serum  is 
injected  intraperitoneally,  according  to  the  technique  of  Neufeld, 
twenty-four  hours  before  injection  of  the  organisms.  On  the  follow- 
ing day,  a  series  of  virulence  controls  is  inoculated  intraperitoneally 
with  appropriate  quantities  of  organisms,  and  the  serum  animals 
are  injected,  intraperitoneally,  with  doses  of  twenty-four-hour  cul- 
tures ranging  from  0.001  to  0.00000001  c.c.  (though  these  quantities 
must  be  adapted  to  the  known  virulence  of  the  strain).  Animals 
that  survive  for  a  period  of  five  days  are  considered  by  Dochez  and 
Avery  as  sufficiently  protected.  The  following  protocol,  taken  from 
the  paper  by  Dochez,  Avery  and  Lancefield,  illustrates  the  method. 

PROTOCOL  I 

In  this  protocol  is  shown  the  titration  of  the  serum  of  a  sheep  immunized  against 
Strain  23.  The  culture  employed  for  infection  was  an  18-hour  broth  culture  of 
No.  S  23,  which  had  received  eighteen  passages  through  white  rats  and  mice.  Each 
mouse  had  received  0.5  c.c.  of  immune  serum  24  hours  previous  to  infection. 


VIRULENCE  CONTROL 


PROTECTIVE  POWER  OF  SERUM  S  23 


Dose  of  Culture, 
c.c. 

Result. 

Dose  of  Culture, 
c.c. 

Result. 

0  001 

S. 

0.0001 

D.  in  4  days 

0.00001 

D.*  in  24  hours  x 

0.000001 

S. 

0.000001 

D.*  in  24  hours 

0.0000001 

S. 

0.0000001 

D.*  in  24  hours 

0.0000001 

S. 

In  the  tables  D.  indicates  died,  S.  survived. 


10-  Dochez,  Avery  and  Lancefield,  Jour.  Exper.  Med.,  30,  1919,  179. 


THE   STREPTOCOCCI  433 

Leucocyte  extracts103  have  been  employed  in  various  forms  of 
streptococcus  infections  of  man,  with  success  in  many  cases.  Favor- 
able results  have  been  obtained  with  these  extracts  in  cases  of  ery- 
sipelas. 

The  agglutinins  found  in  streptococcus  immune  sera  are  usually 
most  active  toward  the  race  of  bacteria  employed  in  the  immunization. 
Other  streptococci  are  also  agglutinated,  but  in  relatively  higher  con- 
centration. While  a  specific  group  reaction  is  useful  in  differentiating 
streptococci  from  other  species,  agglutination  can  not  be  relied  upon 
to  differentiate  individual  streptococci  from  one  another  (Hiss).  It 
has  been  found  that  a  serum  produced  with  a  streptococcus  from  one 
source  contained  a  higher  agglutinating  value  for  some  other,  strep- 
tococcus than  for  the  one  employed  in  its  production.  Agglutinins 
may  be  produced  by  treating  animals  with  dead  as  well  as  with 
the  living  streptococci.  The  technique  of  streptococcus  agglutina- 
tion is  difficult  unless  we  are  dealing  with  strains  which  grow  with 
even  clouding  in  fluid  media.  The  frequent  spontaneous  clumping 
in  broth  cultures  necessitates  the  use  of  a  special  technique.  The 
most  simple  of  these  methods  is  the  one  in  which  calcium-carbonate- 
glucose  broth  is  used  for  cultivation.104  Growing  in  this  medium 
and  thoroughly  shaken  once  a  day,  the  streptococci  are  found  evenly 
divided  in  the  supernatant  fluid  after  the  settling  out  of  the  calcium- 
carbonate  powder. 

This  method,  however,  is  not  often  successful.  In  fact,  a  con- 
stantly reliable  method  for  the  agglutination  of  hemolytic  strepto- 
cocci has  not  yet  been  devised. 

The  method  upon  which  recent  antigenic  study  has  been  based, 
has  depended  upon  the  use  of  a  special  sugar  free  broth  containing 
buffers,  since,  in  acid  cultures,  the  clumping  of  streptococci  is  par- 
ticularly noticeable.  This  method  has  been  developed  particularly 
by  Krumwiede  and  by  Dochez  and  Avery. 

The  special  broth  for  these  purposes  is  made  from  heart  muscle 
which  is  carefully  selected  so  that  fat  is  avoided,  and  instead  of 
using  sodium  chloride,  a  sufficient  quantity  of  a  balanced  phosphate 
mixture  is  added  to  give  the  required  salt  concentration,  and  to 
adjust  the  hydrogen  ion  to  a  P  H  of  7.4.  The  hemolyticus  is  grown 
on  this  for  twenty-four  hours,  at  which  time  it  usually  produces 

103  Hiss,  Jour.  Med.  Res.,  xix,  1908. 

104  7/is.s,  Jour.  Exp.  Med.,  vii,  1905. 


434  PATHOGENIC   MICROORGANISMS 

a  PH  of  7.2.  The  organisms  arc  then  centrifugcd  down,  arc  washed 
once  or  twice  in  the  stock  broth  of  a  PH  of  7.4  prepared  as  above. 
They  are  then  resuspended  in  the  same  medium  in  approximately 
the  original  concentration  of  the  culture,  and  this  suspension  is  used 
for  agglutination.  This  method  has  given  sufficiently  reliable  results, 
and,  although  we  have  tried  a  great  many  other  procedures,  such 
as  grinding  the  organisms  in  salt,  shaking  them  for  long  periods, 
etc.,  we  have  not  been  able  to  obtain  homogeneous  mixtures  better 
adapted  for  agglutination  tests.  For  the  present  we  would  recom- 
mend the  method  of  Dochez  and  Avery  as  perhaps  the  most  useful, 
but  it  cannot  be  regarded  as  entirely  satisfactory  as  yet.  (For 
preparation  of  the  medium  see  chapter  on  media.) 

Passive  therapeutic  immunization  with  polyvalent  antistrepto- 
coccus  serum  has  not  yet  given  reliable  results.  In  view  of  the 
i recent  work  on  pneumococcus  sera  we  believe  it  should  be  subjected 
to  careful  clinical  trial  especially  in  cases  of  spreading  cellulitis  and 
streptococcus  pneumonias  and  empyemas  which  do  not  yield  to  the 
ordinary  forms  of  treatment. 


THE    PROBLEM   OF    MUTATION 

The  many  minor  differences  in  morphology  and  cultural  char- 
acteristics which  have  been  noticed  among  the  streptococci  have 
lead  to  many  assumptions  of  possible  mutations  in  this  group.  It 
is,  of  course,  well  known  that  changes  in  the  length  of  chains  in 
the  production  of  green  color  by  viridans  strains,  and  the  degree 
of  hemolysis  can  be  observed  in  streptococci  grown  in  the  laboratory 
for  a  long  time.  These  changes,  however,  may  4)e  regarded  as 
analogous  to  minor  changes  produced  under  conditions  of  artificial 
cultivation  in  many  other  groups,  such  as  the  typhoid  bacilli  and  others, 
but  do  not  imply  a  fundamental  change  or  true  mutation.  Burger  and 
Wyntenberg105  claimed  that  passage  through  white  mice  converted 
a  capsulated  hemolytic  streptococcus  into  what  they  took  to  be  a 
pneumococcus.  Rosenow106  has  been  the  most  prominent  worker 
along  these  lines,  and  in  1912  claimed  that  he  had  transformed  a 
capsulated  streptococcus  into  a  typical  streptococcus  by  cultivation 

105  Burger  and  Wyntenberg,  Jour.  Infec.  Dis.,  4,  1907,  609. 
™  Rosenow,  Jour.  Infec,  Dis.,  14,  1914,  1. 


THE  PROBLEM   OF  MUTATION  435 

on  agar,  and  later  he  claims  that  by  means  of  animal  passage  he 
had  transformed  a  number  of  viridans  strains  into  pneumococcus, 
and  a  number  of  hemolyticus  strains  into  viridans.  Similar  claims 
have  Been  made  by  Davis.107  It  is  very  difficult  to  comment  upon 
these  results.  Broadhurst108  also  observed  changes  in  fermentation 
reactions  produced  in  streptococci  when  they  were  subjected  to 
growth  in  fresh  milk,  saliva  and  extracts  of  fresh  tissues,  but  she 
draws  conservative  conclusions,  and  changes  such  as  those  noted 
by  her,  might  well  be  minor  ones  in  their  biological  significance. 
The  fundamental  changes  noted  by  Rosenow  particularly  would 
serve  to  disturb  considerably  the  methods  of  classification  now  em- 
ployed. The  subject  is  so  important  that  the  greatest  conservatism 
in  critical  evaluation  of  these  claims  must  be  exercised.  There  are 
many  sources  of  error,  such  as  the  possibility  in  animals  of  injecting 
one  organism  and  getting  another  one  out,  and  the  many  possibilities 
of  contamination,  that  further  work  must  be  done  along  these  lines 
before  judgment  can  be  finally  given.  We,  ourselves,  have  put 
viridans  organisms  into  rabbits  in  open  celloidin  agar  capsules,  by 
a  method  described  by  Raymond  and  ourselves109  and  have,  after 
four  months,  recovered  the  identical  unchanged  organisms  that  we 
put  in,  although  they  had  been  in  the  rabbit  as  long  as  four  months. 
It  is  our  opinion  at  the  present  time  that  such  fundamental  muta- 
tions do  not  take  place,  and  that  it  will  take  a  great  deal  of  very 
careful  and  accurate  work  before  such  claims  can  be  seriously 
considered. 

™  Davis,  Jour.  Infee.  Dis.,  12,  1913,  386. 

108  Broadhurst,  Jour.  Infec.  Dis.,  17,  1915,  277. 

109  Zinsser  and  Raymond,  Transac.  Soc.  Exper.  Med.  and  Biol.,  January,  1921. 


CHAPTER   XXIII 

THE   COMMON   COLD,    THE   PNEUMOCOCCUS   AND  A  CONSIDERATION 
OF    THE    PNEUMONIAS 

THE    COMMON    COLD 

IN  dealing  with  infections  of  the  respiratory  tract  of  man,  it  is 
impossible  to  avoid  referring  briefly  to  a  condition,  which,  though 
mild,  is  perhaps  the  most  common  of  all  such  infections,  is  unques- 
tionably transmitted  from  person  to  person,  and  yet  is  etiologically 
entirely  obscure.  The  condition  is  of  great  importance  because  of 
the  great  loss  of  economic  efficiency  which  wholesale  infection  of  a 
population  with  the  common  cold  entails,  and  because  of  the  fact 
that  the  catarrhal  inflammation  of  the  nose,  throat  and  upper 
bronchii,  which  accompanies  the  cold,  prepares  a  site  for  the  lodg- 
ment and  multiplication  of  influenza  bacilli,  pneumococci,  strepto-. 
cocci,  diphtheria  bacilli,  perhaps  meningococci,  and  other  organisms 
that  may  lead  to  more  serious  disease.  Also,  the  sneezing,  coughing 
and  expectoration  of  individuals  suffering  from  colds  results  in  the 
promiscuous  distribution  of  bacteria  lodged,  by  chance,  in  the  respir- 
atory passages  of  such  people.  Carriers  of  virulent  organisms  of 
various  kinds,  such  as  diphtheria  bacilli,  meningococci,  streptococci, 
pneumococcus  types  I,  II,  and  III,  etc.,  begin,  in  the  course  of 
their  colds,  to  distribute  the  virulent  organisms  they  carry  to  others. 
They  not  only  spread  the  virus  that  has  given  them  the  "cold,"  but 
scatter  a  spray  which  contains  the  virulent  organisms  to  which  they 
themselves  are  immune,  and,  therefore,  not  only  directly  infect  sus- 
ceptible contacts,  but  transmit  to  them  directly  a  condition  which 
will  make  it  possible  for  these  virulent  organisms  to  lodge  in  their 
mucuous  membranes,  and  perhaps  cause  the  secondary  more  serious 
diseases.  A  circuit  of  carrier  distribution  is  so  started,  and  it  has 
been  variously  proved  that  during  the  colder  months  of  the  year, 
when  colds  abound,  the  carrier  rate  of  all  respiratory  diseases 
increases.  If  we  consider  that  in  any  large  group  of  people  there 
may  be  three  or  more  per  cent  of  meningococcus  carriers,  similar 

436 


THE  COMMON 'COLD  437 

percentages  of  pneumococcus,  streptococcus  carriers,  a  fraction  of 
a  per  cent  of  virulent  diphtheria  carriers,  and  that  in  this  group  a 
number  of  the  carriers  begin  to  cough,  spit  and  hawk,  the  percentage 
of  all  these  will  go  up,  and  susceptible  contacts  will  not  only  contract 
the  cold,  but  will  get  the  specific  disease.  These  facts  have  been 
noted  in  the  case  of  meningococci  by  Bassett-Smith,1  for  diphtheria 
bacilli  by  Moss,2  and  the  writer  has  frequently  noted  this  with  pneu- 
mococci  and  streptococci  in  army  camps. 

It  is  thus  seen  that  the  common  cold  offers  indirect  problems  of 
great  importance,  and  that  the  fluctuations  of  the  bacterial  flora  of 
the  mouth,  nose  and  throat,  incident  to  the  common  colds,  are  of 
interest  entirely  apart  from  the  problems  of  specific  etiology  of  the 
cold  itself. 

As  to  the  etiology  of  so-called  colds,  little  is  definitely  known.  It 
is  uncertain  whether  it  is  caused  by  a  single  or  by  a  variety  of 
infectious  agents.  Though  streptococci,  pneumococci,  and  numerous 
other  organisms  have  been  described  as  possible  causative  agents,  to 
none  of  these  can  etiological  importance  be  conclusively  attached. 
Kruse  3  in  1914  published  work  which  suggests  that  the  cold  may 
be  due  to  a  filterable  virus.  He  succeeded  in  transmitting  the  condi- 
tion to  human  beings  with  filtered  mucus.  Similar  work  by  Foster  4 
in  1917  has  seemed  to  bear  out  Kruse 's  contention.  Foster  went  so 
far  as  to  believe  that  he  had  cultivated  the  filterable  virus  by  the 
anaerobic  methods  utilized  by  Noguchi  and  others  in  treponema 
cultivation,  and  described  in  another  place.  An  experiment  done  by 
Hopkins  in  our  laboratory  suggests  similarly  the  participation  of  a 
filterable  virus,  but  it  must  be  remembered  that  experiments  during 
the  season  of  colds  on  human  beings  are  fraught  with  many  possi- 
bilities of  error,  and  many  recent  workers  have  failed  to  obtain 
positive  results.  The  etiology  of  the  common  cold,  therefore,  is  in 
doubt,  and  awaits  further  elucidation. 

Meanwhile,  the  sanitary  importance  of  the  condition  must  not  be 
underestimated,  and  the  principles  of  prevention  are  prefectly  plain, 
although  they  offer  almost  insuperable  difficulties  to  successful 
enforcement. 


1  Bassett-Smith,  Lancet,  194,  1918,  290. 

-Moss,  Guthrie  and  Gelicn,  Transac.   1511)   Internal..  Congress,  Hygiene,  Wash- 
ington, 1913.  < 

3  Kruse,  Munch.  Med.  Woch.,  61,  1914,  1547. 

4  Foster,  Jour.  Infec.  Dis.,  21,  1917,  451. 


438  PATHOGENIC   MICROORGANISMS 

The  striking  power  of  the  cold  is  probably  not  very  great,  and 
direct  or  indirect  contact,  relatively  close  in  time  and  space,  seems 
to  be  necessary  for  transmission.  The  gravity  of  the  malady  itself 
is  so  slight  that  it  is  difficult  to  impress  upon  individuals  the 
necessity  for  care,  and  the  very  grave  influence  upon  general 
respiratory  epidemiology  cannot  be  made  clear  to  those  not  profes- 
sionally interested. 

It  is  our  belief  that  general  rigid  attention  to  the  prevention  of 
colds  in  schools,  hospitals,  military  units,  and  other  closely  associated 
groups  of  people  would  indirectly  exert  a  very  considerable  effect 
upon  the  general  respiratory  sick  rate. 

Prevention  depends  upon  impressing  these  facts  upon  the  public 
and  laying  stress  upon  the  great  danger  of  severe  secondary  disease. 

The  avoidance  of  close  contact,  sleeping  in  the  same  beds,  avoid- 
ance of  the  kissing  of  children,  protecting  companions  from  contam- 
ination by  coughing  and  sneezing,  disinfection  of  handkerchiefs,  etc., 
may  prevent  the  disease  from  going  through  families  as  is  so  often 
the  case. 

Children  in  the  initial  stages  of  severe  colds  should  be  excluded 
from  school  for  a  day  or  two.  Periodical  disinfection  of  nose  and 
throat  with  20  per  cent  argyrol  solution  has  a  definite  effect  on  pre- 
venting transmission,  in  our  opinion. 

Of  especial  importance .  is  the  attention  to  colds  during  the 
existence  of  epidemics  of  diphtheria,  measles,  poliomyelitis,  menin- 
gitis, influenza,  and  the  epidemic  pneumonias  that  may  take  place  in 
army  camps.  Under  such  conditions  the  common  cold  may  be  the 
main  "catalysing  agent,"  as  it  were,  which  keeps  the  more  serious 
disease  active.  At  such  times  people  with  increased  mucous  secre- 
tions who  cough,  spit  and  distribute  mucus  with  handkerchiefs  and 
hands,  are  a  sufficiently  grave  menace  to  call  for  rigid  public  health 
measures.  That  these  cannot  be  successfully  enforced  in  the  general 
population  of  cities,  seems  plain.  But  they  can  be  controlled  in 
factories,  schools,  military  organizations,  hospitals,  asylums,  and 
perhaps,  under  certain  conditions,  in  places  of  amusement,  and 
innumerable  ideal  opportunities  for  spread  can  thus  be  eliminated. 


THE  PNEUMOCOCCUS  AND  PNEUMONIA 

The  opinion  that  lobar  pneumonia  is  an  infectious  disease  was 
held  by  many  far-sighted  clinicians  long  before  the  actual  bacterio- 


THE   PNEUMOCOCCU8  AND   PNEUMONIA  439 

logical  facts  had  been  ascertained.  This  idea,  so  well  founded  upon 
the  nature  of  the  clinical  course  of  the  disease,  with  its  violent  onset 
and  equally  rapid  defervescence,  led  many  of  the  earlier  bacteriol- 
ogists to  make  it  the  subject  of  their  investigations — a  subject  made 
doubly  difficult  by  the  abundant  bacterial  flora  found  normally  in 
the  upper  respiratory  passages,  and  by  the  fact,  which  is  now  recog- 
nized, that  lobar  and  other  pneumonias  are  by  no  means  always 
caused  by  one  and  the  some  microorganisms. 

Cocci  of  various  descriptions  and  cultural  characteristics  were 
isolated  from  penumonia  cases  by  Klebs,5  Koch,fi  Giinther,7  Talamon,8 
and  many  others,  which,  however,  owing  to  the  insufficient  differen- 
tial methods  at  the  command  of  these  investigators,  cannot  positively 
be  identified  with  the  microorganism  now  known  to  us  as  Diplo- 
coccus  pneumoniae  or  the  pneumococcus.  Although  thus  unsuccessful 
as  to  their  initial  object,  these  early  investigations  were  by  no  means 
futile,  in  that  they  gave  valuable  information  regarding  the  manifold 
bacterial  factors  involved  in  acute  pulmonary  disease  and  inci- 
dentally led  to  the  discovery  by  Friedlander 9  of  B.  mucosus 
capsulatus. 

A  tabulation  is  given  in  the  monograph  published  on  acute  lobar 
pneumonia  by  Avery,  Chickering,  Cole  and  Dochez.10  "  Among  529 
cases  diagnosed  from  the  clinical  and  pathologic  features  as  acute 
lobar  pneumonia,  the  following  were  the  etiologic  agents  concerned: 

Diplococcus   pneumoniae    454 

Friedlander 's  bacillus    3 

Bacillus  influenzae    6 

Streptococcus  pyogenes    7 

Streptococcus    mucosus    1 

Staphylococcus  aureus    3 

Cases  of  mixed  infection  with  combinations  of  Staphylococcus  aureus,  Fried- 
lander 's  bacillus,  B.  influenzae,  Streptococcus  pyogenes,  and  Strepto- 
coccus viridans  6 

Undetermined  (Most  of  them  occurring  before  accurate  methods  for  de- 
termining the  etiologic  agent  had  been  devised 49 


Total 529 

5  Klebs,  Arch,  f .  exp.  Path.,  1873. 

6  Koch,  Mitt.  a.  d.  kais,  Gesundheitsamt,  Bd.  1. 

7  Giinther,  Deut.  mod.  Woch.,  1882. 

8  Talamon,  Progr.  med.,  1883. 

9  Friedlander,  Virchow's  Arch.,  Ixxxvii. 

10  Avery,  Chickering,  Cole  and  Dochez,  Monograph  of  The  Rockefellei  Inst.  for 
Med.  Res.,  No.  7,  Oct.  16,  1917. 


440 


PATHOGENIC   MICROORGANISMS 


Communications  upon  lance-shaped  cocci  found  in  saliva,  and 
capable  of  producing  septicemia  in  rabbits,  were  published  almost 
simultaneously  by  Sternberg ll  and  by  Pasteur 12  in  1880.  These 
workers,  beyond  reasonable  doubt,  were  dealing  with  the  true  pneu- 
mococcus,  but  did  not  in  any  way  associate  the  microorganisms  they 
described  with  lobar  pneumonia.  The  solution  of  this  problem  was 
reserved  for  the  labors  of  A.  Frankel 13  and  Weichselbaum  14  who 
published  their  results,  independently  of  each  other,  in  1886,  demon- 
strating beyond  question  that  the  pneumococcm  is  the  etiological 
factor  in  a  large  majority  of  cases  of  lobar  pneumonia. 

Recent  studies  by  Neufeld  and  Haendel,  and  in  this  country  by 
Cole  and  his  coworkers  at  the  Rockefeller  Hospital,  have  shown  that 
in  the  pneumococci  we  are  dealing  not  with  a  single  organism,  but 
with  a  group.  In  this  grouping  three  specific  types  have  been 
described,  named  respectively,  types  I,  II,  and  III,  and  a  heterogene- 
ous collection  of  organisms  classified  for  convenience  together  as 
type  IV.  In  a  subsequent  section,  under  the  main  heading  of 
"Immunity  and  Specific  Therapy,"  this  phase  of  the  pneumococcus 

problem  is  discussed  at   greater 
length. 

Morphology  and  Staining. — 
The  morphology  of  the  pneumo- 
coccus is,  in  general,  one  of  the 
most  valuable  guides  to  its  iden- 
tification. 

When  typical,  the  pneumo- 
coccus is  a  rather  large,  lancet- 
shaped  coccus,  occurring  in  pairs, 
and  surrounded  by  a  definite 
and  often  wide  capsule,  which 
usually  includes  the  two  ap- 
proximated cocci  without  a  defi- 
nite indentation  opposite  their 
lines  of  division.  The  pneumo- 
cocci may,  however,  occur  singly 
or  in  short  chains,  and  even  fairly  long  chains  are  not  infrequently 


FIG.  48. —  PNEUMOCOCCI,  GROWN  ON 
LOEFFLER'S  SERUM.  (Capsule  stain 
by  gentian-violet-potassium-carbonate 
method.) 


11  Sternberg,  Nat.  Board  of  Health  Bull.,  1881. 
^-Pasteur,  Bull,  de  Tacad.  de  med.,  1881. 

13  A.  FrdnTcel,  Zeit.  f.  klin.  Med.,  x,  1886. 

14  Wcichselbaum,  Med.  Jahrbueher,  Wien,  1886. 


THE  PNEUMOCOCCUS  AND   PNEUMONIA  441 

met  with  under  artificial  cultural  conditions.  This  may  be  chiefly 
due  to  the  cultural  conditions  or  may  be  a  prominent  characteristic 
of  certain  strains.  Apparently  the  capsules  of  organisms  making 
up  the  chains  are  continuous ;  wavy  indentations  are  usually  present, 
however,  in  the  capsule  of  chains,  and  at  times  distinct  divisions 
are  observed. 

The  chief  variations  from  the  typical  morphology  consist  either  in 
the  assumption  of  a  more  distinctly  spherical  coccus  type,  or  in  an 
elongation  approximating  the  bacillary  form.  Under  certain  condi- 
tions of  artificial  cultivation  a  distinct  flattening  of  the  organisms, 
particularly  of  those  making  up  chains,  may  be  seen,  and  even  the 
impression  of  a  longitudinal  line  of  division,  characteristic  of  many 
streptococcus  cultures,  is  not  infrequently  gained. 

The  capsules  under  certain  conditions,  especially  in  artificial 
media,  may  be  absent  or  not  demonstrable,  and  in  certain  strains 
capsules  apparently  may  not  be  present  under  any  conditions. 
Practically  any  of  the  described  variations  may  dominate  one  and 
the  same  culture  under  different  or  even  apparently  the  same  con- 
ditions of  cultivation,  and  all  grades  may  occur  in  capsule  develop- 
ment, from  its  typical  formation  through  all  variations,  to  its  total 
and  apparently  permanent  absence. 

The  presence  or  absence  of  capsules  depends,  to  a  large  extent, 
upon  the  previous  environment  of  the  pneumococci  under  observa- 
tion. The  most  favorable  conditions  for  the  development  or  preser- 
vation of  the  pneumococcus  capsule  are  found  in  the  body  fluids  of 
man  and  animals  suffering  from  pneumococcus  infection.  For 
instance,  capsules  may  be  demonstrated  with  ease  by  the  usual 
capsule-staining  methods  in  the  blood,  serum,  and  inflammatory 
exudate  of  the  infected  rabbit  and  white  mouse.  Capsules  may  be 
equally  well  marked  in  the  fresh  sputum  of  pneumonia  patients, 
especially  in  the  early  stages  of  the  disease  and  in  the  exudate  accom- 
panying such  pneumococcus  infections  as  meningitis,  otitis  media, 
and  empyema.  In  sputum  and  the  exudates  of  various  localized 
infections,  the  organisms  are,  however,  frequently  degenerated  or 
under  chemical  conditions  unfavorable  for  capsule  staining,  and 
satisfactory  results  are  not  then  easily  obtained.  The  same  is  often 
true  of  the  scrapings  from  lungs  of  patients  dead  of  pneumonia, 
even  in  the  stage  of  red  hepatization. 

In  artificial  cultivation,  if  the  nutrient  medium  is  not  milk  or 
does  not  contain  serum,  capsules  cannot  usually  be  demonstrated  by 


442  PATHOGENIC   MICROORGANISMS 

the  ordinary  methods  of  preparing  and  staining.  Capsules  may, 
however,  with  much  regularity  be  demonstrated  on  pneumococci,  in 
agar,  broth,  or  on  almost  all,  if  not  all,  artificial  media,  irrespective 
of  the  length  of  time  the  organisms  have  been  under  artificial  culti- 
vation if  beef  or  rabbit  serum  is  used  as  the  diluent,  when  they  are 
spread  on  the  cover-glass  for  staining.15 

The  pneumococcus  is  non-motile  and  possesses  no  flagella.  Spores 
are  not  formed.  Swollen  and  irregular  involution  forms  are  common 
in  cultures  more  than  a  day  old. 

The  pneumococcus  is  stained  readily  with  all  the  usual  aqueous 
anilin  dyes.  Stained  by  the  method  of  Gram,  it  is  not  decolorized. 
Special  methods  of  staining  have  been  devised  for  demonstration  of 
the  capsule.  The  ones  most  generally  used  are  the  glacial  acetic-acid 
method  of  Welch 16  and  the  copper-sulphate  method  of  Hiss.17 

Huntoon's  capsule  stain  described  in  the  section  on  staining 
is  the  easiest  one  to  apply  successfully  on  material  from  cul- 
tures. It  is  not  adapted  to  use  on  pathological  material.  More 
recently  Buerger 18  has  devised  a  more  complicated  method  for 
staining  capsules,  for  which  he  claims  differential  value.  (For 
methods  see  section  on  Technique,  p.  117.) 

For  simple  staining  of  pneumococci  in  tissue  sections,  the  Gram- 
Weigert  technique  is  excellent.  For  demonstration  of  the  capsules 
in  tissue  sections,  Wadsworth  19  has  described  a  simple  method. 

Cultivation. — The  pneumococcus  being  more  strictly  parasitic 
than  many  other  bacteria,  presents  greater  difficulties  in  its  cultiva- 
tion. On  meat-extract  media  growth  does  not  take  place  with 
regularity.  On  those  media,  however,  which  have  beef  or  veal  infu- 
sion for  their  basis,  growth  can  be  obtained  with  considerable 
regularity,  although  such  growth  may  be  sparse  and  delicate. 

Growth  takes  place  most  regularly  at  a  temperature  of  37.5°  C. 
Development  does  not  usually  occur  below  25°  nor  above  41°  C.20 
At  ordinary  room  temperature,  18-22°  C.,  the  temperature  used  for 
gelatin  cultivation,  growth  either  does  not  take  place  at  all  or  is 
exceedingly  slow  and  unenergetic. 


15  Hiss,  Cent,  f .  Bakt.,  xxxi,  1902 ;  Jour.  Exp.  Med.,  vi,  1905. 
19  Welch,  Johns  Hopk.  Hosp.  Bull.,  xiii,  1892. 
"Hiss,  Cent.  f.  Bakt.,  xxxi,  1902;  Jour.  Exp.  Med.,  vi,  1905. 
13  Buer fjer,  Medical  News,  Ixxxviii,  1904. 

19  Wadsworth,  ' '  Studies  by  the  Pupils  of  W.  T.  Sedgwick, ' '  Chicago,  1896. 

20  A.  Frankel,  Dent.  med.  Woch.,  xiii,  1886. 


THE  PNEUMOCOCCUS  AND   PNEUMONIA  443 

Aerobic  and  anaerobic  conditions  both  permit  the  growth  of 
pneumococcus,  there  being  very  little  difference  in  speed  or  extent 
of  growth  along  the  course  of  deep  stab  cultures  in  favorable  media. 
The  most  favorable  reaction  of  media  for  the  cultivation  of  this 
microorganism  is  a  PH  of  7.6  to  7.8.  Slight  acidity,  however,  if  not 
exceeding  eight-tenths  per  cent,  does  not  materially  hamper  develop- 
ment. 

The  broth  or  agar  basis  for  pneumococcus  media  must  be  care- 
fully made,  both  in  regard  to  nutrient  contents  and  reaction.  Ordi- 
nary meat  extract  media  are  not  usually  rich  enough,  and  even 
carelessly  made  meat  infusion  broth  may  fail  to  grow  pneumococci. 
In  our  laboratory  we  have  come  to  use  the  "hormone"  broth  and 
agar  media  almost  entirely  for  pneumococcus  and  streptococcus 
work. 

The  growth  of  pneumococci  on  all  media  may  be  considerably 
enhanced  by  the  addition  to  these  media  of  animal  or  human  serum 
or  whole  blood.  Additional  substances  which,  among  others, 
unquestionably  have  a  favorable  influence  upon  pneumococcus 
growth,  are  glucose,  nutrose,  and  glycerin.  The  addition  of  the 
latter  substances  to  the  media,  however,  probably  because  of  acid 
formation,  hastens  the  death  of  pneumococcus  cultures.  An  increase 
of  the  amount  of  pepton  used  for  the  preparation  of  media  is  desir- 
able for  the  cultivation  of  this  microorganism;  two  to  four  per  cent 
of  pepton  may  be  found  advantageous. 

Transfer  of  recently  isolated  penumococcus  from  broth  culture 
to  broth  culture  necessitates  rather  heavy  inoculation,  since  frequent 
failure  is  experienced  when  only  a  loopful  or  so  is  transferred.  Cole 
mentions  that  about  0.1  c.c.  should  be  transferred  for  every  5  c.c.  of 
broth. 

In  suitable  nutrient  broth,  growth  is  rapid,  and  within  twenty- 
four  hours  leads  to  slight  clouding  of  the  fluid.  This  clouding,  as  a 
rule,  eventually  disappears  as  the  microorganisms,  sinking  to  the 
bottom  of  the  tube  or  disintegrating,  leave  the  fluid  more  or  less  clear. 
In  broth,  pneumococci  have  a  tendency  to  form  short  chains.  When 
glucose  has  been  added  to  the  broth,  growth  is  more  rapid  and  profuse, 
but  considerable  acid  formation  causes  the  cultures  to  die  out  rapidly. 
It  is  possible,  however,  to  employ  glucose  as  a  growth-enhancing  ele- 
ment in  broth  cultures  without  interfering  with  the  viability  of  the 
cultures  by  adding  small  quantities  (one  per  cent)  •  of  sterile,  powdered 
calcium  carbonate.  This  method  of  cultivation  in  broth  is  especially 


444  PATHOGENIC  MICROORGANISMS 

adapted  to  the  production  of  mass  cultures  for  purposes  of  immuniza- 
tion or  agglutination.21  The  addition  of  ascitic  fluid  or  blood  serum 
to  broth,  in  the  proportion  of  one  to  three,  makes  an  extremely  favor- 
able medium  in  which  growth  is  rapid  and  profuse. 

Upon  agar  plates,  pneumococcus  growth  is  not  unlike  that  of  strep- 
tococcus. The  colonies  are  small,  round,  and  slightly  more  transparent 
than  those  of  the  streptococci.  They  appear  more  moist  than  strepto- 
coccus colonies  and  often  are  more  flat.  Microscopically  examined,  the 
colonies  are  finely  granular,  with  dark  centers  and  slightly  corrugated 
lighter-colored  peripheral  areas.  Under  high  magnification  no  such 
intertwining  convolutions  can  be  seen  as  those  noticed  under  similar 
magnification  in  streptococcus  cultures.  The  addition  of  animal 
albumin  to  agar  results  in  the  more  rapid  development,  larger  size, 
and  deeper  opacity  of  the  colonies. 

Agar  stab  cultures  show  growth  within  twenty-four  to  thirty-six 
hours,  which  takes  place  with  equal  thickness  along  the  entire  course 
of  the  stab.  There  is  nothing  distinctive  in  these  cultures  to  differen- 
tiate them  from  similar  streptococcus  cultures. 

In  gelatin  plate  and  stab  cultures  at  22°  C.,  growth,  as  a  rule,  does 
not  take  place.  This,  however,  is  not  true  of  all  races  of  penumococci. 
Occasionally  strains  are  met  with  which  will  grow  fairly  abundantly  in 
gelatin  at  a  temperature  of  22°  C.  When  the  gelatin  is  rendered 
sufficiently  firm  to  bear  25°  to  26°  C.  without  melting,  growth  appears 
slowly  and  sparsely  as  minute,  grayish-white,  transparent  colonies. 
The  gelatin  is  not  liquefied. 

Growth  upon  milk  is  profuse,  resulting  in  the  production  of  acid 
and  coagulation  of  the  medium.  Kaces  are  encountered  in  which  this 
is  suppressed  and  coagulation  in  milk  is  absent  or  long  delayed. 

Upon  potato,  thin,  gray,  moist  growth  occurs,  hardly  visible  and 
indistinguishable  from  an  increased  moisture  on  the  surface  of  the 
medium.  This  is  not  a  favorable  medium. 

Upon  Loeffler's  coagulated  ~blood'serum,  the  pneumococcus  develops 
into  moist,  watery,  discrete  colonies  which  tend  to  disappear  by  a 
drying  out  of  the  colonies  after  some  days,  differing  in  this  from 
streptococcus  colonies,  which,  though  also  discrete,  are  usually  more 
opaque  and  whiter  in  appearance  than  those  of  the  pneumococcus 
and  remain  unchanged  for  a  longer  time.  This  medium,  as  will  be 
seen,  is  useful  in  differentiating  pneumococci  from  the  so-called 
Streptococcus  mucosus. 


21  Hiss,  Jour.  Exp.  Med.,  vii,  1905. 


THE  PNEUMOCOCCUS  Ai^D  PNEUMONIA  445 

Upon  mixtures  of  whole  rabbit's  blood  and  agar,  the  pneumococcus 
grows  well,  and  forms,  after  four  or  five  days,  thick,  black  surface 
colonies,  not  unlike  sun  blisters  on  red  paint.  These  colonies  are  easily 
distinguished  from  those  of  streptococci,  and  are  of  considerable 
differential  value.22 

Pneumococcus  colonies  on  blood  plates  may  cause  a  slight  halo  of 
hemolysis  and  methemoglobin  formation  with  a  zone  of  greenish  color 
about  the  colony  after  48  hours  or  longer  in  the  incubator. 

The  hemolysin  formation  by  pneumococcus  is  slight,  but  quite 
definite.  It  occurs  late,  rarely  sooner  than  48  hours,  but  is  mentioned 
because  errors  of  diagnosis  through  ignorance  of  this  might  occur. 
Cole23  has  particularly  studied  the  hemotoxins  of  this  organism. 

Guarnieri 24  has  recommended  a  medium  with  a  pepton-beef- 
infusion  basis  rendered  semisolid  by  mixtures  of  agar  and  gelatin. 
A  modification  of  this  medium  has  been  described  by  Welch  25  and  has 
been  much  employed.  Cultivation  within  eggs  and  upon  egg  media26 
has  been  used.  Wadsworth27  has  recommended  a  medium  composed 
of  ascitic  fluid  to  which  agar  has  been  added — sufficient  to  give  a  soft, 
jelly-like  consistency.  He  observed  prolonged  viability  and  the  preser- 
vation of  the  virulence  on  this  medium. 

For  the  purpose  of  differentiating  pneumococci  from  streptococci, 
Hiss28  devised  a  medium  of  beef  serum  one  part,  and  distilled  water 
two  parts,  to  which  is  added  one  per  cent  of  inulin  (c.  p.),  and  enough 
litmus  to  render  the  medium  a  clear,  transparent  blue.  By  fermenta- 
tion of  the  inulin  the  pneumococcus  acidifies  this  mixture,  causing 
coagulation  of  the  serum.  Streptococci  do  not  ferment  inulin. 

Since  inulin  fermentation  is  a  very  important  differential  character- 
istic of  the  pneumococcus,  it  is  necessary  to  say  a  few  words  about 
the  irregularity  with  which  it  occurs.  Occasionally,  failure  to  ferment 
inulin  is  due  to  the  fact  that  the  particular  strain  of  pneumococcus 
does  not  grow  well  in  the  inulin  medium  made  up  by  the  older  method 
of  Hiss.  The  addition  of  one  per  cent  of  pepton  to  this  medium  has 
been  suggested  by  Burger  and  is  a  distinct  improvement.  It  may 

22  Hiss,  loc.  cit. 

23  Cole,  Jour.  Exp.  Mc<L,  1914,  20,  346  and  363. 
s*  Giumticri,  Alt.  dell'  Aead.  di  Roma,  1883. 
-•Wr1ch,  Johns  Hopk.  Hosp.  Bull.,  iii,  1892. 
2SSV/«ro,  Kiv.  d'lgiene,  1894. 

27  Wadswortli,  Proc.  N.  Y.  Path.  Soc.,  1903. 

28  Hiss,  Jour.  Exp.  Med.,  vi,  1905. 


446  PATHOGENIC   MICROORGANISMS 

happen,  however,  that  occasional  strains  will  react  irregularly  on 
different  preparations  of  inulin.  These  irregularities  must  be  taken 
into  consideration  when  this  test  is  used  for  differential  purposes. 

Isolation. — For  the  isolation  of  pneumococci  from  mixed  cultures 
or  from  material  containing  other  species,  such  as  sputum,  surface 
smears  of  the  material  are  made  upon  plates  of  neutral  glucose-agar, 
glucose-serum-agar,  or  blood  agar.  According  to  the  number  of 
bacteria  present  in  the  infected  material,  it  may  be  smeared  directly 
upon  the  plate,  or  diluted  with  sterile  broth  before  planting.  After 
incubation  for  twenty-four  hours,  the  pneumococcus  colonies  are 
easily  differentiated  from  all  but  those  of  streptococcus.  With 
practice,  however,  they  may  be  distinguished  from  these  also,  by 
their  smoother  edges  and  greater  transparency  and  flatness. 

The  easiest  way  to  isolate  pneumococci  from  mixed  culture  and 
especially  from  material  from  patients  like  sputum,  pulmonary 
exudate,  etc.,  is  injection  ^nto  white  mice.  When  sputum  is  used 
the  sputum  should  be  washed  by  gently  rinsing  in  successive  watch 
glasses  or  pipette  plates  containing  salt  solution  or  broth.  It  can 
be  injected  directly  into  a  white  mouse,  intraperitoneally,  or,  if  very 
stringy  or  dry,  can  be  rubbed  in  a  mortar  with  a  little  broth  before 
injection.  Great  care  must  be  taken  not  to  inject  too  much  material. 
The  details  of  this  method  are  given  in  connection  with  clinical 
considerations  in  a  subsequent  paragraph.  If  virulent  pneumococci 
are  present,  death  will  occur  within  24  hours,  or  thereabouts.  Pneu- 
mococci will  be  found  in  pure  culture  in  the  heart's  blood  and  in 
large  numbers  in  the  peritoneal  exudate. 

PNEUMOCOCCUS  TYPES. — As  stated  above,  Neufeld  and  Haendel 29 
in  1910  found  that  pneumococci  were  by  no  means  all  alike,  sero- 
logically.  Although  all  the  true  pneumococci  have  morphological 
and  cultural  characteristics  which  would  appear  to  classify  them  as 
a  single  species,  it  was  found  that  within  this  apparently  homologous 
group  there  were  sharp  serological  differentiations.  Dochez  and 
Gillespie  30  not  only  confirmed  the  work  of  Neufeld  and  Haendal,  but 
made  a  careful  study  of  pneumococcus  types  as  they  occurred  in 
America,  both  by  agglutination  reactions  and  by  protection  tests  on 
mice.  They  isolated  a  large  number  of  pneumococcus  strains,  and 
immunized  animals  with  them.  The  sera  of  these  animals  woro  now 


29  Neufeld  and  Ha,endel,  Arb.  a.  <1.  k.  Gsndhtsamte,  34,  1910,  293. 
and  Gillespie,  Jour,  of  the  A.  M.  A.,  61,  1913,  727. 


THE   PNEUMOCOCCUS    AND   PNEUMONIA  447 

examined  for  cross-agglutination  with  the  various  strains  and  for 
their  protective  powers  on  mice.  It  was  found  that  the  pneumococci 
they  studied  fell  into  very  sharp  classes.  The  surprising  thing  about 
their  results  is  that  the  distinctions  between  the  types  seem  to  be  so 
sharp  that  very  little  "group"  reaction  occurs. 

Their  classification,  which  has  been  many  times  confirmed  and 
which  may  be  accepted  as  representing,  in  a  tentative  way,  the  con- 
ditions existing  in  the  United  States  at  any  rate,  divides  pneumo- 
cocci into  four  main  types,  numbered  accordingly,  I,  II,  III,  and  IV. 

Types  I  and  II  are  morphologically  and  culturally  typical  pneumo- 
cocci, and  represent  well  circumscribed  species,  sharply  classifiable  by 
the  fact  that  members  of  type  I  agglutinate  only,  and  are  protected 
against  only,  by  type  I  serum,  and  members  of  type  II  will  react 
similarly  only  with  type  II  serum.  Type  III  represents  what  was 
formerly  spoken  of  as  the  Streptococcus  Mucosus,  but  which  is 
included  in  the  pneumococci  because  of  its  inulin  fermentation,  bile 
solubility,  and  pathogenic  properties  which  are  quite  similar  to  those 
of  the  pneumococci. 

By  type  IV  is  meant  a  heterogeneous  group  which  comprises  all  of 
the  true  pneumococci  which  cannot  be  serologically  placed  into  the 
other  three  types.  This  fourth  group  is  merely  a  matter  of  con- 
venience of  nomenclature,  since  few  of  these  organisms  are  related  to 
each  other.  They  repreesnt  a  sort  of  attic  into  which  unclassifiable 
pneumococci  have  been  thrown  for  the  present,  until  they  can  be  sorted 
out.  Olmstead  31  has  studied  a  considerable  number  of  so-called  type 
IV  organisms,  and  has  found  that  smaller  subgroups  could  be  estab- 
lished by  serological  classification.  But  so  many  different  subgroups 
were  found  that,  for  the  present,  no  practical  results  have  come  out  of 
these  attempts  to  systematize  group  IV.  The  difficulties  in  this  group 
are  similar  to  those  encountered  in  connection  with  streptococcus 
viridans. 

Although  type  II  is  a  very  sharply  defined  variety,  there  are  cer- 
tain subgroups  within  type  II,  studied  particularly  by  Avery.32  They 
consist  of  atypical  members  of  group  II  which  are  agglutinated  by 
type  II  serum,  with  diminished  intensity.  All  of  them  will  be 
agglutinated  with  the  concentrated  serum,  but  when  dilutions  of  1 
to  20  or  over  are  used,  they  fail  to  agglutinate.  Avery  has  defined 
three  such  subgroups  of  type  II  which  he  calls  subgroups  Ila,  II&, 

31  Olmstead,  Jour,  of  Immunol.,  2,  1917,  425. 

32  Avery,  Jour,  of  Exper.  Med.,  22,  ]915,  804. 


448  PATHOGENIC  MICROORGANISMS 

and  llx.  All  of  these  are  proven  members  of  type  II  because  they  all 
agglutinate  in  concentrated  type  II  serum.  All  but  llx  are  protected 
against  by  type  II  serum.  Absorption  of  type  II  serum  with  a  type  II 
organism  removes  the  antibodies  for  all  the  subgroups.  Absorption 
of  the  anti-pneumococcus  type  II  serum  with  any  one  member  of  a 
subgroup  removes  only  the  antibodies  for  that  particular  subgroup. 

That  the  three  subgroups  are  specifically  different  from  each  other 
and  from  type  II,  is  determined  by  Avery  by  the  fact  that  any  given 
subgroup  is  not  agglutinated  by  antisera  made  with  organisms  of  the 
other  two  subgroups.  The  same  is  true  of  protection  reactions.  Also, 
no  one  of  them  absorbs  the  antibodies  for  the  other  subgroups  from 
serum. 

Subgroups  "A"  and  "B"  have  immunity  reactions  identical,  each 
within  its  respective  group.  Subgroup  IIx,  however,  consists  of  heter- 
ogeneous strains  in  which  each  strain  seems  to  produce  its  character 
istic  antibodies  specific  only  for  the  strain  used. 

In  addition  to  these  types,  there  are  unquestionably  a  number  of 
other  types  in  different  parts  of  the  world.  Thus,  Lister  33  working  in 
South  Africa,  found  three  homogeneous  types,  A,  B,  C,  his  types  C 
and  B  corresponding  to  types  I  and  II  of  the  American  classification, 
respectively;  his  type  A  being  not  so  far  identified  with  any  of  our 
American  types. 

The  importance  of  this  discovery  of  the  serological  pneumococcus 
group  in  serum  therapy  is  obvious,  and  its  bearing  on  epidemiology  is 
dealt  with  in  another  section. 

Pneumococcus  (streptococcus)  Mucosus. — First  definitely  de- 
scribed by  Howard  and  Perkins  34  in  1901,  and  subsequently  carefully 
studied  by  Schottmuller,35  who  isolated  it  from  cases  of  parametritis, 
peritonitis,  meningitis,  and  phlebitis.  It  has  since  been  described  by 
many  as  the  incitant  of  lobar  pneumonia  and  of  a  variety  of  other 
lesions  and  as  often  an  apparently  harmless  inhabitant  of  the  normal 
mouth.  Morphologically,  though  showing  a  marked  tendency  to  form 
chains,  on  solid  media  it  often  appears  in  the  diplococcus  form.  It  is 
enclosed  in  an  extensive  capsule,  which  appears  with  much  regularity 
and  persistence.  Though  very  similar  in  appearance,  therefore,  to 
pneumococci,  these  bacteria  do  not  appear  in  the  typical  lancet  shape. 
Upon  solid  media  they  show  a  tendency  to  grow  in  transparent  moist 

33  Lister,  Pub.  South  African  Inst.  for  Med.  Res.,  1916,  No.  8. 
"Howard  and  Perkins,  Jour.  Med.  Res.,  1901,  N.  S.,  i. 
85  ScJiott mutter,  Munch,    med.  Woch.,  xxi,  1903. 


THE  PNEUMOCOCCUS  AND  PNEUMONIA  449 

masses.  The  regularity  with  which  this  microorganism  ferments 
inulin  medium,  make  it  probable  that  it  is  more  accurate  to  place  it 
with  the  group  of  pneumococci  than  with  that  of  streptococci.30 

Most  of  the  organisms  of  this  group  show  the  common  character- 
istics of  the  pneumococci  and  are  soluble  in  bile.  Occasional  strains, 
such  as  one  studied  by  Dochez  and  Gillespie,  neither  ferment  inulin 
nor  are  bile  soluble.  Rarely,  too,  does  it  cause  hemolysis.  From  the 
various  studies  carried  out  upon  this  group  it  must  be  concluded  that 
while  perfectly  distinct  in  its  formation  of  a  heavy  mucoid  colony  and 
capsulation,  this  group  is  more  closely  related  to  the  pneumococci  than 
to  the  true  streptococci.  As  would  be  expected  from  its  capsulation, 
its  virulence  is  very  powerful  and  serum  reactions  are  not  easily  car- 
ried out.  A  further  discussion  of  the  immune  serum  reactions  with 
this  organism  is  included  in  this  chapter,  page  462. 

Resistance. — On  artificial  media,  the  viability  of  the  pneumo- 
coccus  is  not  great.  Cultures  upon  agar  or  bouillon  should  be  trans- 
planted every  third  or  fourth  day,  if  the  cultures  are  kept  within  an 
incubator.  In  all  media  in  which  rapid  acid  formation  takes  place, 
such  as  glucose  media,  the  death  of  cultures  may  occur  more  rapidly. 
In  media  containing  albumin  and  of  a  proper  reaction,  preservation 
for  one  or  even  two  weeks  is  possible.  The  longer  the  particular  race 
has  been  kept  upon  artificial  media,  the  more  profuse  is  its  growth, 
and  the  greater  its  viability,  both  qualities  going  hand  in  hand  with 
diminishing  parasitism.  The  length  of  life  may  be  much  increased  by 
preservation  at  low  temperature,  in  the  dark,  and  by  the  exclusion  of 
air.  In  calcium  carbonate  broth  and  kept  in  the  ice-chest,  cultures 
may  often  remain  alive  for  months. 

Neufeld  has  succeeded  in  keeping  pneumococci  alive  and  virulent, 
by  taking  out  the  spleens  of  mice  dead  of  pneumococcus  infection  and 
preserving  them  in  a  Petri  dish  in  a  desiccator,  in  the  dark  and  cold. 
In  this  way,  the  organisms  can  be  cultivated  from  the  spleen,  and  will 
be  found  virulent  for  longer  periods  than  in  culture  media.  The  best 
way  to  get  such  cultures  back  is  by  injecting  a  suspension  of  the 
desiccated  spleen,  in  broth,  into  a  mouse,  and  recovering  the  pneumo- 
coccus from  the  heart's  blood. 

In  sputum  the  viability  of  pneumococci  seems  to  exceed  that 
observed  in  culture.  The  studies  of  (luariiieri,37  Bordoni-Uffreduzzi,38 

367/is-s,  Jour.  Exp.,  Mcd.,  1905;  Buergcr,  Cent.  f.  Bakt.,  I,  xli,  1906. 
37  Guarnicri,  Atta  della  R.  Acad.  Med.  di  Eoma  iv,  1888. 
88  Bordoni- Uffredussi,  Arch.  p.  1.  Sc.  med.  xv,  1891. 


450  PATHOGENIC  MICROORGANISMS 

and  others  have  shown  that  pneumococci  slowly  dried  in  sputum  may 
remain  alive  and  virulent  for  1  to  4  months,  when  protected  from 
light ;  and  as  long  as  nineteen  days  when  exposed  to  diffused  light  at 
room  temperature.  Experiments  hy  Ottolenghi 39  have  confirmed  these 
results;  the  virulence  seems,  in  Ottolenghi 's  experiments,  to  have 
become  considerably  attenuated  before  death  of  the  cocci.  Recent 
studies  by  Wood,40  whose  attention  was  focused  chiefly  upon  pneumo- 
coccus viability  in  finely  divided  sputum — in  a  condition  in  which 
inhalation  transmission  would  be  possible — have  shown  that  pneumo- 
cocci survive  for  only  about  one  and  one-half  hours,  under  ordinary 
conditions  of  light  and  temperature.  Exposed  to  strong  sunlight, 
pneumococci  die  off  within  an  hour. 

Low  temperatures  slightly  above  zero  are  conducive  to  the  pro- 
longation of  life  and  the  preservation  of  virulence. 

The  resistance  of  the  pneumococcus  to  heat  is  low,  52°  C.  destroy- 
ing it  in  ten  minutes.41  To  germicidal  agents,  carbolic  acid,  bichlorid 
of  mercury,  permanganate  of  potassium,  etc.,  the  pneumococcus  is 
sensitive,  being  destroyed  by  weak  solutions  after  short  exposures. 

The  disinfection  of  sputum,  difficult  because  of  the  protective  coat- 
ing of  the  secretions  about  the  bacteria,  has  been  recently  studied  by 
Wadsworth.42  The  conclusions  reached  by  this  writer  indicate  that 
pneumococci  in  exudates  are  most  rapidly  destroyed  by  twenty  per 
cent  alcohol,  other  and  stronger  disinfectants  being  less  efficient,  prob- 
ably because  of  slighter  powers  of  diffusion. 

Differentiation  of  Pneumococcus  from  Streptococcus. — Pneumo- 
cocci and  streptococci  which  do  not  differ  in  morphology  from  their 
classic  types  can  usually  be  differentiated  from  each  other  and  identi- 
fied by  their  morphological  characters  without  difficulty;  but  it  is 
equally  true  that  certain  cultures  of  these  organisms,  either  at  the  time 
of  their  isolation  or  after  cultivation  on  artificial  media,  approach  the 
type  of  the  other  so  closely  that  it  may  be  impossible  to  identify  them 
by  their  morphology  alone.  When  such  morphological  variations  occur 
there  are  no  constant  cultural  or  pathogenic  characters  as  yet  demon- 
strated which  distinguish  between  these  organisms. 

This  lack  of  distinct  cultural  differences  between  pneumococci  and 
streptococci  has  not  infrequently  led  to  confusion,  and  that  uncer- 

39  Ottolenghi,  Cent,  f .  Bakt.,  xxv,  1889. 

40  Wood,  Jour.  Exp.  Med.,  vii,  1905. 
"Sternberg,  Cent.  f.  Bakt.,  xii,  1891. 

42  Wadsworth,  Jour.  Inf.  Diseases,  iii,  1906. 


THE  PNEUMOCOCCUS  AND  PNEUMONIA  451 

tainty  should  exist  and  mistakes  be  made  in  identification  is  not  sur- 
prising when  one  considers  the  characters  usually  depended  upon  to 
distinguish  pneumococci  from  streptococci.  Chief  among  these,  as  has 
just  been  implied,  are  the  morphological  features  which  are,  in  the 
case  of  pneumococci,  a  slightly  lancet  or  elongated  form  rather  than 
the  more  typical  coccus  form  characteristic  of  the  streptococci,  and  an 
arrangement  of  such  cocci  in  pairs  rather  than  in  chains;  added  to 
these  features  is  the  possession  of  a  more  or  less  well-defined  capsule. 
All  of  these  characters  are  subject  to  variation  or  may  be  absent.  Com- 
pared with  the  morphological,  the  cultural  characters  are  variable  and 
of  minor  importance.  The  pneumococcus  colonies  on  coagulated  blood 
serum  and  on  agar  are  moister  and  flatter,  and  the  freshly  isolated 
pneumococcus  is  usually  unable  to  develop  readily  or  at  all  on  gelatin 
at  below  22°  C. 

The  distinctness  of  the  capsule  of  the  pneumococcus  in  the  body 
fluids  of  man  and  animals  and  on  blood  serum,  milk,  or  serum  agar, 
has  been  depended  upon  as  the  chief  distinguishing  and  diagnostic 
character.  Nevertheless,  instances  have  been  reported  of  distinct  cap- 
sule formation  by  organisms  which  had  either  been  previously  identi- 
fied as  Streptococcus  pyogenes,  or  at  the  time  of  the  isolation  could  not 
be  definitely  identified  as  belonging  to  this  group  or  to  the  pneumo- 
cocci, but  were  considered  intermediate  in  character.43 

43  Brief  Description  of  Organisms  Eeported  as  Capsulated  Streptococci. — Bordet 
(Bordet,  Ann.  de  1'inst.  Pasteur,  1897,  xi,  p.  177),  working  with  an  organism 
previously  identified  as  Streptococcus  pyogenes,  described  such  capsule  formation 
occurring  in  the  peritoneal  exudate  of  infected  rabbits. 

Schuetz'  (Schuetz,  Cent.  f.  Bakt.,  Kef.  1,  1887,  p.  393)  Diplokokkus  der 
Brustseuche  der  Pferde,  Poels  and  Nolen's  (Poels  und  Nolen,  Ford.  d.  Med.,  iv, 
1886,  p.  217)  streptococcus  of  contagious  pneumonia  of  cattle,  and  especially 
the  organism  described  by  Bonome  (Bonome,  Ziegler's  Beit.,  viii,  1890,  p.  377) 
as  Streptococcus  der  meningitis  cerebrospinalis  epidemica,  may  all  be  looked  upon 
as  organisms  differentiated  on  insecure  grounds  from  either  pneumococcus  or 
streptococcus.  The  first  two  of  these  organisms,  however,  are  said  to  be  decolorized 
by  Gram's  method,  and  as  suggested  by  Frosch  and  Kolle  (Frosch  und  Kolle, 
Fliigge's  ' ' Mikro-organis., ' '  ii,  1896,  p.  161),  in  the  case  of  Schuetz'  organism 
may  belong  to  a  group  intermediate  between  Fraenkel's  diplococcus  and  the 
chicken-cholera  group. 

Tavel  and  Krumbein  (Tavel  und  Krumbein,  Cent.  f.  Bakt.,  xviii,  1895,  p.  547) 
describe  a  streptococcus  with  a  capsule,  which  was  isolated  from  a  small  abscess 
on  the  finger  of  a  child.  Capsules  were  also  present  in  the  artificial  cultures, 
and  although  ordinarily  remaining  uncolored,  could  be  stained  by  Loeffler's 
flagella  stain.  This  organism  was  said  to  be  differentiated  from  Fraenkel's 
diplococcus  and  also  in  general  from  streptococcus  (pyogenes)  by  a  rapid  and 


452  PATHOGENIC  MICROORGANISMS 

There  are  occasions,  then,  both  within  the  animal  body  and  in  arti- 
ficial cultivations,   when  it  is  practically  impossible  to   distinguish 

rich  growth  on  gelatin,  agar,  and  potato.  A  pellicle  was  formed  on  broth.  The 
organisms  forming  this  pellicle  had  capsules,  but  those  in  the  deeper  portions 
of  the  broth  generally  lacked  it. 

In  1897  Binaghi  (Binaghi,  Cent.  f.  Bakt.,  xxii,  1897,  p.  273)  described  a 
capsulated  streptococcus  isolated  from  a  guinea-pig  dead  of  a  spontaneous  peri- 
bronchitis  and  multiple  pulmonary  abscesses.  In  the  pus  were  found  some  diplo- 
cocci  and  short  chains  (four  to  six)  surrounded  by  a  cupsule,  shown  by  staining 
with  carbol  fuchsin.  This  organism  he  proposes  to  call  Streptococcus  capsulatus. 
Le  Koy  des  Barres  and  Weinberg  in  1899  (Le  Eoy  des  Barres  et  Weinberg, 
Arch.  d.  med.  exper.,  xi,  1899,  p.  399)  published  an  account  of  a  streptococcus 
with  a  capsule.  This  was  isolated  from  a  man  who  had  apparently  been  infected 
from  a  horse  which  had  died  of  an  acute  intestinal  disorder.  The  patient  neglected 
the  infection  and  died.  Diplococci  and  short  chains  furnished  with  a  capsule 
were  found  in  the  subcutaneous  tissue  at  the  area  of  infection.  The  blood,  liver, 
and  spleen  also  contained  these  organisms.  The  capsule  in  all  the  preparations 
remained  uncolored,  but  the  authors  say  that  its  existence  was  not  to  be  doubted. 
Ascitic  broth  inoculated  from  the  peritoneal  exudate  of  a  rabbit  dying  from  the 
infection  gave  streptococci  in  extremely  long  chains  and  surrounded  by  capsules. 
These  were  not  so  distinct  as  in  the  case  of  the  organisms  in  the  original  smear 
preparations.  All  fluid  media  (bouillon,  milk,  and  ascitic  broth)  were  said  to 
be  strongly  acid  after  twenty-four  hours.  These  authors  report  that  Achard  and 
Marmorek  have  assured  them  that  they  have  seen  capsulated  streptococci,  and 
that  Marmorek  showed  them  some  preparations  in  which  one  of  his  streptococci 
presented  the  same  characters  as  that  isolated  by  them. 

Although  Le  Eoy  des  Barres  and  Weinberg  have  used  the  term  encapsulated, 
they  believe  that  it  would  perhaps  be  more  prudent  to  call  their  organism  strepto- 
coque  aureole,  since  they  were  not  able  to  define  this  capsule  by  staining  it. 

t  Howard  and  Perkins  (Howard  and  Perkins,  Jour.  Med.  Res.,  1901,  iv,  p.  163) 
have  lately  described  an  organism,  probably  of  the  foregoing  type,  which  was 
present  in  a  tubo-ovarian  abscess  and  in  the  peritoneal  exudate,  the  blood,  and 
some  of  the  organs  of  a  woman  dying  in  the  Lakeside  Hospital,  Cleveland,  Ohio. 
The  organisms  were  biscuit -shaped  cocci  in  pairs,  usually  arranged  in  chains  of 
four,  six,  eight,  or  twenty  elements,  and  surrounded  by  a  wide  and  sharply 
staining  capsule.  In  the  artificial  cultures  special  capsule  stains,  it  was  noted, 
failed  to  stain  any  definite  area,  but  numerous  small  deeply  stained  granules 
were  to  be  seen  within  the  halo,  especially  near  its  outer  border.  Howard  and 
Perkins  propose  for  the  group  composed  of  the  streptococci  of  Bonome,  Binaghi, 
and  their  own  organism,  the  name  Streptococcus  mucosus.  Streptococci  isolated 
from  cases  of  epidemic  sore-throat  have  also  shown  capsules  (p.  421). 

Reference  to  the  original  descriptions  of  these  various  capsulated  streptococci 
will  show  that,  with  the  exception  of  a  rather  poorly  staining  capsule,  the 
majority  of  these  organisms  are  separated  from  the  typical  Streptococcus  pyogenes 
or  from  the  pneumococcus  by  exceedingly  slight  and  unstable  morphological  and 
cultural  characters.  This  is  true  of  the  difference  in  their  pathogenic  action 
in  animals. 


THE  PNEUMOCOCCUS  AND  PNEUMONIA  453 

definitely  between  some  races  of  pneumococci  and  races  of  streptococci. 
This  difficulty  is  especially  heightened  when  the  piieumococcus  has 
become  non-virulent,  and  at  the  same  time  no  very  typical  morphology 
or  capsule  formation  is  to  be  determined  and  a  tendency  to  chain- 
formation  is  marked.  Cultures  of  pneumococci  in  such  condition  can- 
not readily  be  distinguished  morphologically  from  streptococcus  cul- 
tures. 

Under  these  circumstances  recourse  must  be  had  to  a  careful  bio- 
logical study  of  the  organism  in  question.  The  following  are  the 
criteria  mainly  relied  upon  at  present  for  the  differentiation  of  these 
two  groups. 

Pneumococci  ferment  inulin,  if  cultivated  in  inulin-serum-water 
medium.  Acid  formation  from  the  inulin  results  within  two  days  or 
more  in  coagulation  of  the  serum  and  reddening  of  the  litmus.  Strep- 
tococci, because  of  their  inability  to  attack  the  inulin,  leave  the 
medium  unchanged.44 

Cultivated  on  whole-blood-agar,  streptococci  usually  cause  hemoly- 
sis,  pneumococci  usually  do  not.45  In  contradistinction  to  Strepto- 
coccus viridans  which  does  not  hemolyze,  pneumococci  have  a  tendency 
on  these  media  to  form  the  black,  dry,  paint-blister  colonies.46 

Neufeld,47  in  1900,  noticed  that  normal  rabbits'  bile  added  in  quan- 
tities of  0.1  c.c.  to  each  one  or  two  cubic  centimeters  of  a  pneumococcus 
broth  culture  caused  lysis  of  the  bacteria,  rendering  the  culture  fluid 
transparent  and  clear.  This  does  not  occur  with  streptococci,  and  has 
been  used  to  differentiate  the  two  species.  According  to  Libman  and 
Rosenthal,48  great  reliance  may  be  placed  upon  this  method. 

The  most  convenient  reagent  for  use  in  the  Neufeld  bile  test  is  a 
10  per  cent  solution  of  sodium  taurochlorate  in  physiological  salt  solu- 
tion. This  should  be  sterilized  or  kept  on  ice.  One-tenth  volume  of 
such  a  solution  produces  prompt  lysis  in  a  broth  culture  of  pneumo- 
cocci. 

Decisive  differential  importance  may  be  attached  to  the  agglutina- 
tions of  these  microorganisms  in  immune  sera  (see  p.  462). 

The  permanency  of  the  various  types  in  the  pneumococcus-strepto- 


44  Hiss,  Cent,  f .  Bakt.,  xxxi,  1902 ;  Jour.  Exp.  Med.,  vi,  1905. 

45  Schottmuller,  Miinch.  med.  Woch. 

46  Hiss,  Jour.  Exp.  Med.,  vii,  1905. 

47  Neufeld,  Zeit.  f.  Hyg.,   1901. 

48  Libman  and  Bosenthal,  Proc.  N.  Y.  Path.  Soe.,  March,  1908. 


454  PATHOGENIC   MICROORGANISMS 

coccus  group  is  still  open  to  question.  E.  C.  Rosenow  49  has  recently 
reported  that  he  has  transmuted  typical  pneumococci  into  typical 
hemolytic  streptococci  by  methods  which  he  has  not  as  yet  fully 
described,  but  among  which  were  animal  passage,  growth  in  symbiosis 
with  bacillus  subtilis,  and  growth  in  an  atmosphere  of  oxygen.  The 
pneumococci  when  first  altered  took  on  the  characteristics  of  the 
streptococcus  viridans,  later  of  the  so-called  streptococcus  rheumaticus, 
and  finally  of  streptococcus  hemolyticus.  Together  with  cultural 
characteristics  the  pathogenicity  of  these  various  strains  for  rabbits 
changed.  The  pneumococcus  produced  acute  sepsis,  the  streptococcus 
viridans  caused  endocarditis,  the  streptococcus  rheumaticus  peri- 
articular  or  serous  arthritis,  and  hemolyticus  suppurative  arthritis. 
In  intermediate  stage  the  organisms  quite  regularly  caused  myositis. 
Although  he  was  able  to  transmute  these  types  one  into  the  other  in 
both  directions,  Rosenow  believes  that  the  cultural  characteristics  of 
each  type  correspond  to  a  fairly  definite  type  of  pathogenicity  in  ani- 
mals and  man.  This  work  has  not  as  yet  appeared  in  detail  and  has 
not  been  confirmed. 

Toxic  Products  of  the  Pneumococcus. — Our  knowledge  of  pneu- 
mococcus poisons  is  still  very  imperfect.  Attempts  to  obtain  soluble 
toxins  by  the  filtration  of  cultures  have  been  practically  unsuccessful. 
G.  and  F.  Klemperer,50  Mennes,51  Pane,52  Foa  and'  Carbone,53  and 
others  failed  to  obtain  pneumococcus  filtrates  of  any  degree  of  toxicity, 
though  working  with  highly  virulent  strains.  The  feeble  toxin  so 
obtained  produced  no  antitoxin. 

The  general  failure  to  procure  strong  soluble  poisons  from  cultures, 
gives  weight  to  the  assumption  that  the  most  potent  toxic  products  of 
pneumococci  are  in  the  nature  of  endotoxins  and  closely  bound  to  the 
cell-bodies  themselves.  This  assumption  is  borne  out  by  the  more 
recent  experiments  of  Macfadyen.54  This  author  obtained  acutely 
poisonous  substances  from  pneumococci  by  trituration  of  the  organ- 
isms after  freezing,  and  extracting  them  with  a  one  1 :1,000  caustic 
potash  solution.  With  the  filtrates  of  these  extracts  he  was  able  to 
cause  rapid  death  in  rabbits  and  guinea-pigs  by  the  use  of  doses  not 

™Eosenow,  J.  A.  M.  A.,  1913,  Ixi,  2007. 

50  G.  and  F.  Klemperer,  Berl.  klin.  Woch.,  xxxiv  and  xxxv,  1891. 
"Mennes,  Zeit.  f.  Hyg.,  xxv,  1897. 

KPane,  Eif.  med.,  xxi,  1898. 

53  .Foa  und  Carbone,  Cent.  f.  Bakt.,  x,  1899. 

51  Macfadyen,  Brit.  Med.  Jour.,  ii,  1906. 


THE   PNEUMOCOCCUS  AND   PNEUMONIA  455 

exceeding  0.5  to  1  c.c.  He  found,  furthermore,  a  striking  parallelism 
between  the  degree  of  toxicity  and  the  virulence  of  the  extracted  cul- 
ture. Cole,55  too,  in  recent  studies,  inclines  to  the  belief  that  the 
poisons  of  the  pneumococcus  are  in  the  nature  of  endotoxins  and  has 
produced  toxic  substances  by  salt  solution  and  bile  extraction  of  the 
organisms. 

Cole56  states  that  when  the  cells  of  pneumococci  are  dissolved  with 
bile  salts  the  solution  becomes  hemolytic  and  toxic.  This  hemolytic 
substance  is  easily  destroyed  by  heat,  by  tryptic  digestion,  and  is 
partially  lost  in  nitration  through  Berkefeld  niters.  He  believes  that 
there  is  some  relationship  between  the  virulence  of  the  organisms  and 
these  substances.  An  interesting  fact,  furthermore,  is  that  the  choles- 
trin  inhibits  such  toxic  effects,  but  anti-pneumococcus  serum  is  only 
partially  effective.  ,  Cole  differentiates  this  endocellular  hemotoxin 
from  another  substance  that  converts  hemoglobin  into  methemo- 
globin.  The  latter  substance  is  produced  only  by  the  living  cells,  and 
depends  upon  the  presence  of  oxygen. 

In  addition  to  these  substances,  there  are  very  soluble  materials 
that  appear  in  culture  media  in  which  the  pneumococcus  is  grown, 
during  the  life  of  the  organisms,  and  which  can  be  detected  by  specific 
precipitin  reactions.  These  substances,  as  we  shall  see,  have  been 
found  by  Avery  and  Dochez  57  to  be  present  in  the  blood  and  urine  of 
pneumonia  patients.  It  is  questionable  whether  or  not  they  exert 
toxic  action  in  the  infected  body. 

Virulence  and  Pathogenicity. — The  virulence  of  pneumococci  is 
subject  to  much  variation,  depending  upon  the  length  of  time  during 
which  it  has  been  cultivated.  It  has  been  mentioned  above  that 
under  conditions  such  as  those  prevailing  in  dried  sputum  or  blood 
the  virulence  of  pneumococci  may  be  preserved  for  several  weeks. 
Ordinarily,  the  virulence  diminishes  as  the  cocci  adapt  themselves 
to  life  upon  artificial  media.  Upon  media  containing  animal  albumin, 
such  as  ascitic  fluid  or  blood  agar,  this  attenuation  is  less  rapid  than 
upon  the  simple  meat-infusion  preparations. 

The  maintenance  of  virulence,  according  to  Kirkbride,58  is  greatly 
aided  by  making  transfers  from  broth  to  broth  at  intervals  not 

55  Cole,  Harvey  Lecture,  N.  Y.,  Dec.,  1913. 

56  Cote,  Jour,  of  Exper.  Med.,  16,  1912,  644. 

"Avery  and  Doclicz,  Proc.  Soc.  Exper.  Med.  and  Biol.,   14,  1016,  126. 
M  Kirldride,  Paper  read  before  the  Amer.  Assoc.   Pathol.   and  Bacter.,   New 
York,  April,  1917. 


456  PATHOGENIC   MICROORGANISMS 

longer  than  eight  hours.  Apparently  cultures  grown  for  as  long 
as  twenty-four  hours  diminish  in  virulence  much  more  rapidly.  To 
some  extent  this  observation  is  in  keeping  with  Chesney's59  state- 
ment that  growth  is  best  obtained  when  the  transfers  are  made  from 
broth  at  the  period  of  maximum  growth  rate. 

Swift  has  recently  succeeded  in  keeping  organisms  of  the  pneu- 
mococcus  and  streptococcus  varieties  alive  and  virulent  for  a  long 
time  by  centrifugating  broth  cultures,  taking  off  the  supernatant 
fluid,  and  drying  the  residue  in  a  frozen  condition  in  vacuo.  This 
material  can  be  kept  for  a  very  long  time  without  death  of  the 
bacteria,  and  without  appreciable  loss  of  virulence. 

In  the  blood  of  rabbits  dead  of  a  pneumococcus  infection,  taken 
directly  into  sterilized  tubes,  sealed  and  kept  in  the  dark,  Foa60  has 
been  able  to  preserve  the  virulence  of  pneumococci  for  as  long  as 
forty-five  days.  Preservation  in  the  spleen  of  animals  dead  of  pneu- 
mococcus infection,  as  practiced  by  Neufeld,  has  been  mentioned 
above.  Whether  or  not  the  virulence  of  pneumococci  is  attenuated 
or  enhanced  by  sojourn  within  the  human  body  during  disease  is 
uncertain.  The  attenuation  of  virulent  pneumococci  on  artificial 
media  may  be  hastened,  according  to  Frankel,61  by  cultivation  of 
the  organism  at  or  above  a  temperature  of  41°  C. 

The  virulence  of  attenuated  cultures  may  be  rapidly  enhanced 
by  passage  of  the  organisms  through  the  bodies  of  susceptible 
animals.  The  virulence  of  strains  may  be  so  enhanced  that  one 
one-millionth  of  a  c.c.  will  kill  a  mouse. 

Among  the  domestic  animals  white  mice  and  rabbits  are  most 
susceptible.  Guinea-pigs,  dogs,  rats,  and  cats  are  much  more  resis- 
tant. Guinea-pigs  can  be  given  astonishingly  large  doses  of  pneu- 
mococci without  injury.  Birds  are  practically  immune.  Kyes  who 
has  studied  pneumococcus  infection  in  birds  particularly  has  shown 
that  the  fixed  tissue  cells  of  the  liver,  spleen  and  lungs  destroy  the 
organisms  by  prompt  and  effective  phagocytosis.  His  attempts  at 
treating  pneumonia  with  immune  chicken  serum,  suggested  by  these 
observations,  will  be  referred,  to  below. 

The  results  of  pneumococcus  inoculation  into  susceptible  animals 
vary  according  to  the  size  of  the  dose,  the  virulence  of  the  introduced 
bacteria,  the  mode  of  administration,  and  the  susceptibility  of  the 


mChe*ney,  Jour,  of  Exper.  Med.,  24,  3916,  387. 
"Foa,  Ztschr.  f.  Hyg.   iv,   1888. 
"Franlcel,  Deut.  med.  Woch.,  13,  1886. 


THE  PNEUMOCOCCUS  AND  PNEUMONIA  457 

subject  of  the  inoculation.  Subcutaneous  inoculation  of  virulent 
pneumococci  into  mice  and  rabbits  usually  results  in  an  edematous 
exudation  at  the  point  of  inoculation,  which  leads  to  septicemia  and 
death  within  twenty-four  to  seventy-two  or  more  hours.  When  the 
dose  has  been  extremely  small  or  the  culture  unusually  attenuated, 
a  localized  abscess  may  be  the  only  result.  Intravenous  inoculation 
is  usually  more  rapidly  fatal  in  these  animals  than  the  subcutaneous 
method.  Intraperitoneal  inoculation  in  rabbits  results  in  the  forma- 
tion of  a  rapidly  spreading  peritonitis  in  which  the  exudates  are  apt 
to  be  accompanied  by  a  deposit  of  fibrin,  and  to  lack  the  transparent 
red  color  so  often  caused  by  the  hemolyzing  streptococci.  With 
very  virulent  strains,  these  differences  are  less  marked.  In  almost 
all  of  these  infections  death  is  preceded  by  septicemia  and  the  micro- 
organisms can  be  recovered  from  the  heart's  blood  of  the  victims. 

The  production  in  animals  of  lesions  comparable  to  the  lobar 
pneumonia  of  human  subjects  has  long  been  the  aim  of  many  in- 
vestigators. Wadsworth,62  recognizing  that  such  lesions  probably 
depended  upon  the  partial  immunity  which  enabled  the  infected 
subjects  to  localize  the  pneumococcus  processes  in  the  lungs  after 
infection  by  way  of  the  respiratory  passages,  succeeded  in  producing 
typical  lobar  pneumonia  in  rabbits  by  partially  immunizing  these 
animals  and  inoculating  them  intratracheally  with  pneumococci  of 
varying  virulence.  Lamar  and  Meltzer63  produced  lobar  pneumonia 
in  dogs  in  1912  by  injecting  cultures  in  the  bronchi  and  blowing 
them  into  the  finer  bronchioles  with  air.  Similar  experiments  have 
been  made  by  Winternitz  and  Hirschfelder.64 

The  most  striking  parallelism  between  experimental  animal  in- 
fection and  pneumonia  as  it  occurs  in  man  has  recently  been  obtained 
by  Cecil  and  Blake.  Using  Macaccus  and  other  species  of  monkey, 
they  injected  small  amounts,  0.1  to  0.2  c.c.  of  virulent  pneumococcus 
cultures  directly  into  the  trachea  of  these  animals,  and,  after  an 
incubation  time  of  a  day  or  slightly  longer,  they  obtained  typical 
lobar  pneumonias.  This  work  will  be  further  spoken  of  below. 


62  Wadsworth,  Amer.  Jour.  Med.  Sci.,  May,  1904. 

63  Lamar  and  Meltser,  Jour.  Exp.  Med.,  xv,  1912. 

94  Winternitz  and  Hirschfelder,  Jour.  Exp.  Med.,  xvii,  1913. 


458 


PATHOGENIC   MICROORGANISMS 


PNEUMOCOCCUS  INFECTIONS  IN  MAN,  AND  CLINICAL- 
BACTERIOLOGICAL  CONSIDERATIONS 

In  man  the  most  frequent  lesion  produced  by  the  pneumococcus 
is  acute  lobar  pneumonia.  About  90  per  cent  of  all  cases  of  this 
disease  are  caused  by  the  pneumococcus,  the  remainder  being  due 
to  streptococci,  influenza  bacilli,  and  other  organisms,  the  relative 
frequency  of  which,  in  this  disease  has  been  given  in  an  earlier 
section  of  this  chapter.  The  relative  frequency  of  the  various  pneu- 
mococcus types  in  lobar  pneumonia  in  and  about  New  York  is  given 
as  follows  by  Avery,  Chickering,  Cole  and  Dochez,65  from  whose 
work  the  following  table  is  taken. 

TABLE  2— INCIDENCE  OF  TYPES  OF  PNEUMOCOCCUS  IN  LOBAR 

PNEUMONIA 


Type  of  Pneumococcus. 

INCIDENCE 

No.  Cases. 

Per  Cent. 

I         .     

151 
133 
6 
4 
9 
59 
92 

33.3 
29.3 
1.3 
0.9 
2.0 
13.0 
20.3 

II 

Ha  . 

116          .     ;  .  

llx 

m  

IV               

This  table  may  be  contrasted  with  the  following,  also  the  result 
of  work  by  the  above  named  authors,  which  shows  the  distribution 
of  the  different  types  in  the  mouths  of  normal  individuals,  a  point 
which  has  considerable  importance  in  connection  with  the  problem 
of  autoinfection  with  which  we  will  deal  in  greater  detail  in  speaking 
of  the  epidemiology  of  the  disease. 

There  has  been  a  great  deal  of  discussion  concerning  the  route 
by  which  infection  of  the  lung  comes  about  after  the  pneumococcus 
has  entered  a  susceptible  subject.  The  difficulties  experienced  by 
many  observers  in  infecting  animals  by  direct  instillation  of  pneu- 
mococci  into  the  lungs,  have  inclined  many  observers  to  assume  that 


85  Avery,  Checkering,  Cole  and  Dochez,  Monograph  of  the  Eock.  Inst.,  No.  7, 
Oct.  16,  1917. 


PNEUMOCOCCUS  INFECTIONS   IN   MAN 


459 


in  most  pneumonias  infection  of  the  blood,  or  bacteriemia,  precedes 
pulmonary  lodgment.  The  recent  experiments  of  Blake  and  Cecil,66 
however,  have,  it  seems  to  us,  shown  pretty  definitely  that  the  failure 
of  other  observers  to  produce  direct  infection  of  the  lungs  experi- 
menally,  were  due  very  largely  to  failure  to  choose  the  most  favor- 
able animals  for  this  purpose,  and,  therefore,  failing  to  obtain  the 
balance  between  pathogenicity  of  the  organism  and  resistance  of 
the  subject  which  is  necessary  to  determine  the  localization  of  the 
pneumococci  in  the  pulmonary  alveola. 

TABLE  3— DISTRIBUTION  OF  DIFFERENT  TYPES  OF  PNEUMOCCOCUS 
IN  MOUTHS  OF  NORMAL  PERSONS  * 


Type  of  Pneumococcus 

INCIDENCE 

No.  Cases. 

Per  Cent. 

I    .                              ... 

1 

0 

1 

7 
13 
34 
64 

0.8 
0.0 
0.8 
5.8 
11.6 
28.1 
52.9 

II  

Ila  
116 

llx 

III  

IV.  . 

Pneumococcus  present  
Pneumococcus  absent  .    .  . 

116 
181 

297 

Blake  and  Cecil  succeeded  in  producing  various  types  of  pneu- 
monias in  the  lower  monkeys  (Macaccus  Syrichtus,  etc.)  by  injecting 
small  amounts  of  virulent  organisms  directly  into  the  trachea  with 
a  fine  needle.  When  they  injected  0.1  c.c.  of  an  eighteen-hour  broth 
culture  of  virulent  pneumococcus  they  usually  obtained  definite 
symptoms  within  twenty-four  hours,  at  which  time  the  monkeys 
showed  rapid  respiration  and  positive  blood  cultures.  Such  monkeys 
often  died  within  eight  to  twelve  days,  with  typical  pneumonic 
autopsy  findings,  often  with  actual  fibrinous  pleurisy  and  the  clas- 
sical anatomical  changes  of  human  pneumonia.  These  experiments 
seem  to  represent  a  fairly  accurate  analogy  to  the  human  disease. 


66  Blake  and  Cecil,  Jour.  Exper.  Med.,  31,  1920,  403,  455,  599,  518,  657,  685; 
Jour.  Exper.  Med.,  32,  1920,  1,  and  401. 


460  PATHOGENIC   MICROORGANISMS 

Winternitz,  Smith  and  Robinson67  have,  however,  recently  sug- 
gested another  possibility.  They  have  called  attention  to  the  fact 
that  there  is  a  rich  plexus  of  lymphatics  about  the  trachea,  within 
the  submucosa.  They  believe  that  the  nature  of  the  tracheal  and 
bronchial  submucosa,  with  its  ciliated  mucous  epithelium,  renders 
it  extremely  difficult  for  bacteria  and  other  materials  to  reach  the 
lung  by  this  open  route  under  normal  conditions,  and  call  attention 
to  the  great  difficulty  which  has  been  encountered  in  attempts  to 
produce  disease  by  mere  inhalation  without  pulmonary  irritation 
or  injury  of  the  respiratory  tract.  Their  idea  is  that  infection  of 
the  lung  is  accomplished  by  the  entrance  of  the  bacteria  into  the 
lymphatics  surrounding  the  trachea  through  some  injury  to  the 
nmcosa  and  are  then  afforded  a  direct  path  for  infection.  In  experi- 
mental work  either  the  infecting  needle  or  the  catheter  may  produce 
preliminary  injury  and  lymphatic  inoculation.  They  support  their 
contention  by  inoculating  rabbits  both  by  tracheal  injection  with 
a  needle,  and  by  catheters,  and  finding  in  successful  infections  that 
the  lymphatics  referred  to  were  involved. 

According  to  Cole,68  when  pneumonia  is  secondary  to  septicemia 
it  is  usually  of  the  lobular  type.  Experiments  of  Meltzer  seem  to 
indicate  that  infection  is  facilitated  by  closure  of  the  small  bron- 
chioles, and  cold  or  chilling  may  possibly  stimulate  the  mucous 
glands  so  as  to  plug  these. 

In  the  course  of  the  development  of  pneumonia  the  infecting 
organisms  are,  of  course,  located  in  the  pulmonary  alveolae  and  the 
smaller  bronchioles,  and  appear  in  the  sputum.  Since  the  possibility 
of  specific  serum  therapy  which  will  be  dealt  with  in  detail  below 
have  made  it  desirable  not  only  to  determine  whether  the  disease 
is  caused  by  the  pneumococcus,  but  also  have  necessitated  our 
knowing  which  particular  type  is  responsible  in  the  individual  case, 
all  pneumonia  cases  should  be  typed  whenever  possible. 

Typing  from  Sputum. — A  technique  for  this  has  been  developed 
by  the  workers  mentioned  above  and  the  technique  of  Dochez  and 
Avery,  with  no  essential  changes,  but  a  few  additional  remarks, 
is  as  follows:  Since  it  is  important  not  to  confuse  the  lung  invader 
with  adventitious  pneumococci  present  in  the  mouth,  the  collection 
of  the  sputum  is  an  important  part  of  the  technique.  Failure  to 


67  Winternits,  Smith  and  Eobinson,  Bull.  Johns  Hopkins  Hosp.,  31,  1920,  63. 

68  Cole,  Harvey  Lecture,  New  York,  Dec.  13,  1913. 


PNEUMOCOCCUS   INFECTIONS  IN   MAN  461 

exercise  care  in  this  respect  has  lead  to  many  Type  IV  reports 
when  the  pneumonia  was  actually  caused  by  I,  II,  or  III.  The 
special  sputum  for  typing  -should  be  collected  in  a  separate  cup, 
and  not  in  the  general  sputum  cup  into  which  the  patient  has  been 
spitting  all  day.  The  patient  should  rinse  his  mouth  thoroughly 
with  salt  solution,  bicarbonate  of  soda  solution,  or  perhaps  weak 
alcohol,  and  a  specimen  of  sputum  should  be  immediately  obtained 
by  coughing.  It  is  collected  in  a  sterile  Petri  dish  or  clean  cup, 
and  should  not  be  allowed  to  stand  around  in  the  ward  for  any 
length  of  time,  but  should  be  sent  to  the  laboratory,  or  stored  in 
the  ice  chest. 

A  Gram  stain  should  be  made  of  the  sputum  as  a  preliminary 
survey.  A  capsule  stain  may  also  be  made  with  advantage. 

In  the  rare  cases  in  which  mice  are  not  available,  the  sputum 
should  be  plated  upon  blood  agar  plates  for  subsequent  agglutination 
or  single  colonies  by  the  microscopic  method.  But  this  is  not  de- 
sirable, and  mice  can  usually  be  procured.  We  have  found  gray 
mice  quite  as  suitable  as  white  mice,  and  in  outlying  laboratories 
an  extensive  use  of  mouse  traps  will  solve  the  problem. 

As  soon  as  the  sputum  has  been  received  in  the  laboratory  it 
should  be  washed.  A  small  bit  of  sputum,  preferably  from  the 
center  of  a  clump,  is  lifted  with  a  platinum  needle  into  three  or 
four  Petri  dishes  containing  sterile  salt  solution  or  broth,  and  gently 
swabbed  about.  The  thorough  washing  is  extremely  important  for 
clean-cut  results.  Some  times  the  sputum  can  be  directly  sucked 
up  into  a  sterile  syringe.  If  this  cannot  be  done,  it  should  be  ground 
in  a  sterile  mortar  with  about  1  to  2  c.c.  of  sterile  broth  or  salt 
solution  added  gradually.  About  0.5  c.c.  of  this  is  intraperitoneally 
injected  into  a  mouse.  Great  care  should  be  exercised  not  to  inject 
too  much  since  massive  inoculation  may  give  contaminating  organ- 
isms a  chance,  and  many  mice  have  been  wasted  in  this  way.  In 
the  mouse  the  pneumococcus,  if  virulent,  outgrows  most  other  or^ 
ganisms.  However,  occasionally  mice  will  die  of  streptococcus 
infection  and  may  show  streptococcus,  influenza  and  occasionally 
staphylococcus  infection,  but  this  is  relatively  infrequent. 

The  mouse  will  appear  very  sick  or  die  between  ten  and  twenty- 
four  hours.  When  the  mouse  is  either  in  extremis  or  dead  it  should 
be  immediately  autopsied. 

Pin  the  mouse  down  on  a  small  board,  carefully  dissect  off  the 
skin  and,  with  sterile  instruments,  open  the  peritoneum. 


462 


PATHOGENIC   MICROORGANISMS 


Make  a  smear  of  the  peritoneal  exudate  and  stain  by  Gram.  If 
the  pneumococcus  is  pure  in  the  peritoneum  wash  out  the  peritoneal 
exudate  with  a  small  nipple  pipette,  with  about  3  or  4  o.c.  of  sterile 
salt  solution  into  a  centrifuge  tube.  Make  a  smear  from  the  heart's 
blood  and  also  take  a  culture  on  a  blood  agar  slant  from  the  heart's 
blood  with  sterile  instruments. 

Centrifuge  the  exudate  previously  removed  gently  at  low  speed 
for  a  few  moments  to  throw  down  leucocytes  and  larger  particles. 
Remove  the  turbid  supernatant  fluid  which  should  have  the  maxi- 
mum turbidity  of  a  well  grown  broth  culture  of  pneumococei,  into 
another  centrifuge  tube  and  throw  down  the  organisms  at  high 
speed.  Resuspend  the  sediment  in  enough  salt  solution  to  give  a 
turbid  suspension  of  proper  concentration  for  agglutination  reac- 
tions. With  this  material  agglutinations  are  set  up  in  small  tubes 
as  follows:  The  table  presents  the  routine  method  advised  by  the 
workers  mentioned  above. 


TABLE  1— DETERMINATION  OF  PNEUMOCOCCUS  TYPES  BY 
AGGLUTINATION69 


Pneumococcus. 
Suspension,  0.5  c.c. 

Serum  I 
(1  :  20) 
0.5  c.c. 

Serum  II 
(Undiluted) 
0.5  c.c. 

Serum     II 

(1  :  20) 
0.5  c.c. 

Serum  III 

(1  :  5) 
0.5  c.c. 

Type    I 

+  + 

Type  II 

+  + 

+  + 

Sub-groups  Ila,  6,  x  
Type  III  
Type  IV 

- 

+ 

+  + 

Incubation  for  1  hour  at  37°  C. 

Circumstances  may  arise,  as  in  the  recent  war,  where  it  has 
been  practically  impossible  to  obtain  enough  mice  to  carry  out  the 
above  technique  in  all  cases.  In  such  cases  we  ourselves  have  often 
successfully  typed  from  colonies  grown  on  blood  agar  plates  by 
microscopic  methods,  in  the  unsuccessful  cases  having  typed  subse- 
quently from  blood  cultures.  Avery  has  recently  advised  the  use 
of  a  medium  in  which  the  pneumococcus  is  apt  to  outgrow  other 
organisms.  It  depends  upon  the  use  of  the  following  medium : 

To  90  c.c.  of  a  suitable  meat  infusion  broth  of  a  PH  of  7.8,  add 
5  c.c.  of  a  sterile  20  per  cent  solution  of  dextrose  to  bring  it  to  a 


This  table  taken  from  Avery,  Chickering,  Cole  and  Docker,  loc.  cit. 


PNEUMOCOCCUS  INFECTIONS  IN   MAN  463 

concentration  of  1  per  cent  and  5  e.c.  of  defibrinated  rabbit's  blood. 
These  substances,  sterile,  must  be  mixed  in  tubes  containing  about 
5  c.c.  each  and  must  not  be  resterilized  after  mixture. 

The  sputum  is  very  carefully  washed  in  sterile  salt  solution. 
We  prefer  to  do  the  washing  for  this  particular  technique  in  succes- 
sive test  tubes  of  sterile  broth.  After  about  three  washings,  the 
sputum  is  gently  ground  in  the  last  broth  tube  with  a  glass  rod, 
and  about  0.5  to  1  c.c.  of  this  inoculated  into  tubes  of  the  medium 
given  above.  Growth  is  allowed  to  take  place  for  from  five  to  eight 
hours,  and  when  pneumococci  have  appeared  in  large  numbers  in 
the  supernatant  fluid,  the  red  cells  are  thrown  down  by  centrifuga- 
tion,  and  agglutinations  done  on  the  supernatant  fluid  as  above. 

In  all  such  typings  it  must  not  be  forgotten  that  agglutination 
reactions  must  be  accompanied  by  a  bile  test  on  the  materials  used  in 
order  to  make  sure  that  the  organism  is  a  pneumococcus. 

Typing'  by  Protection  Experiment. — The  protection  experiment 
is  not  very  practical  for  clinical  use  where  speed  is  required.  It 
is,  however,  important  that  the  technique  of  specific  mouse  protec- 
tion should  be  well  known  to  the  bacteriologist.  This  technique  will 
be  given  in  greater  detail  in  connection  with  the  determination  of 
the  potency  of  immune  serum  in  a  subsequent  section. 

Typing  by  Precipitins. — In  broth  cultures  the  pneumococcus 
gives  up  an  antigenic  substance  which  is  soluble  to  the  broth  which 
has  been  made  use  of  by  Blake70  for  typing  purposes.  The  method 
is  useful  when  cultures  or  peritoneal  exudates  from  mice  are  too 
heavily  contaminated  for  clear  agglutination  tests.  The  fluid  to  be 
examined,  either  culture  or  peritoneal  exudate,  is  centrifuged  at 
very  high  speed  to  throw  down  all  the  organisms,  and  the  clear 
supernatant  fluid  taken  off  for  precipitation  experiments.  It  is  then 
mixed  in  quantities  of  0.5  c.c.  with  equal  quantities  of  serum  specific 
for  the  three  main  types  of  pneumococci.  Type  I  serum  is  used  in  a 
1-10  dilution,  Type  II,  undiluted,  1-10,  and  Type  III,  1-5.  In  this 
connection  Dochez  and  Avery71  have  made  an  interesting  observation 
which  is  of  great  theoretical  as  well  as  practical  importance.  They 
have  shown  that  a  soluble  antigenic  substance  which  gives  specific 
precipitin  reactions  with  anti-pneumococcus  serum,  appears  in  the 
urine  of  over  60  per  cent  of  pneumonia  cases.  The  substance  may 


70  Klake,  Jour.  Exper.  Med.,  26,  1917,  67. 

71  Inches  and  Avery,  Jour.  Exper.  Med.,  26,  1917,  477. 


464  PATHOGENIC  MICROORGANISMS 

be  excreted  within  the  first  day  after  the  onset  of  the  disease,  and 
may  continue  in  the  urine  during  early  convalescence.  Since  it  is 
almost  always  present  in  septicemia,  it  may  have  some  value  in 
sizing  up  a  case  in  this  way. 

The  substance  can  be  demonstrated  by  direct  precipitation  of 
the  clear  urine  against  specific  pneumococcus  sera.  A  curious  thing 
about  the  substance  is  that  it  can  be  obtained  in  concentrated  urine 
in  the  following  way.  25  c.c.  of  the  urine  is  boiled  with  weak  acetic 
acid  and  the  proteins  removed  by  filtration.  The  filtrate  is  then 
precipitated  with  95  per  cent  alcohol,  rapidly  dried,  and  redissolved 
in  small  amounts  of  salt  solution  up  to  3  c.c.  This  solution  reacts 
specifically  with  the  serum. 

Blood  Cultures  in  Pneumonia. — During  the  course  of  pneumonia, 
pneumococcus  septicemia  is  common.  Frankel72  in  1902  stated  that 
he  believed  in  most,  if  not  all  cases  of  pneumonia  the  organisms  are 
present  in  the  blood  stream  at  some  stage  of  the  disease.  Proschaska, 
in  a  carefully  repeated  culturing  of  ten  unselected  cases,  obtained 
the  organisms  in  every  one  of  them.  The  older  literature,  if  carefully 
reviewed,  shows  positive  blood  cultures  in  about  25  per  cent  of  the 
cases.  In  the  Rockefeller  Hospital  where  systematic  blood  cultures 
were  done,  among  other  things,  it  is  stated  by  Cole  that  in  448 
cases  of  lobar  pneumonia,  the  pneumococci  were  obtained  by  blood 
culture  in  30.3  per  cent.  When  blood  cultures  were  repeatedly  made 
at  frequent  intervals  the  positive  findings  were  obtained  in  50 
per  cent. 

In  taking  blood  cultures  it  is  important  that  plenty  of  blood  is 
taken  and  inoculated.  The  culture  is  taken  from  the  basilic  vein 
with  a  sterile  syringe  as  follows:  At  least  5  or  10  c.c.  of  blood 
should  be  added  to  flasks  of  hormone  glucose  broth,  of  a  PH  of  7.6 
or  7.8,  containing  not  less  than  100  c.c.  of  broth.  Hormone  glucose 
agar  plates  should  at  the  same  time  be  made,  and  graded  quantities 
of  blood  can  be  added  to  successive  plates  in  order  that  one  may 
obtain  a  numerical  estimate  of  the  number  of  organisms  per  c.c. 
Growth  is  often  delayed,  and  no  negative  report  should  be  finally 
turned  in  for  at  least  three  days.  Cole  and  others  have  attached 
great  prognostic  significance  to  blood  cultures.  Cole  believes  that 
the  development  of  a  septicemia  is  of  very  serious  prognostic  sig- 
nificance, and  the  typing  of  the  organisms  from  the  blood  culture 

72  Frankel,  "v.  Leyden  Festschr.,"  1902. 


PNEUMOOOCCTJS  INFECTIONS  IN  MAN  465 

is  important  in  this  respect  since  the  mortality  of  type  II  cases  in 
the  blood  in  his  experience  is  73.4  per  cent,  and  of  type  III  cases 
in  the  blood,  100  per  cent,  whereas,  in  type  IV  cases  a  mortality  of 
only  52.3  per  cent,  and  the  low  percentage,  26  per  cent,  in  type  I 
blood  culture  cases,  he  attributes  to  the  effect  of  serum  treatment. 

Aside  from  lobar  and  lobular  pneumonia,  the  pneumococcus  may 
cause  a  number  of  other  types  of  infection  in  human  beings  either 
subsequent  to  a  preliminary  pulmonary  infection,  or  primary  in 
nature. 

The  most  common  complications  of  pneumococcus  infection  of 
the  lung  are  empyemia,  endocarditis,  and  pericarditis,  meningitis 
and  arthritis.  Meningitis  may  occur  as  a  primary  disease  especially 
in  children  without  previous  traceable  pneumonia.  The  same  may 
be  said  of  arthritis. 

Empyema  was  a  very  frequent  and  fatal  complication  of  the 
war  pneumonias,  and  pneumococci  can  be  easily  obtained  by  ordinary 
cultural  methods  from  puncture  fluid.  It  has  recently  been  sug- 
gested that  empyema  is  more  apt  to  follow  in  cases  which  have  been 
treated  with  serum,  perhaps  because  of  its  effect  in  localizing  the 
infection.  This  point  has  not  been  settled,  but  it  would  seem  to 
us  that  the  only  manner  in  which  such  a  result  could  eventuate 
would  be  by  just  such  localizing  effect,  and  this  would  mean  that  had 
not  the  serum  localized  the  infection  the  outcome  might  have  been 
fatal,  a  consideration  which  all  the  more  persuades  us  of  the  wisdom 
of  treating  type  I  cases  with  serum  whenever  possible.  Pneumo- 
coccus meningitis,  whether  primary  or  secondary,  is  a  very  fatal 
disease  from  which  recovery  is  rare.  Direct  serum  treatment  should 
always  be  tried  if  it  is  a  type  I  case,  and  intravenous  treatment 
to  forestall  a  possible  septicemia  should  also  be  applied. 

Pneumococcus  peritonitis  occurs  particularly  in  children. 

The  pneumococcus  also  causes  a  very  severe  form  of  corneal 
ulceration  which  presents  great  difficulties  to  successful  therapy. 
The  bacteriologist  confronted  with  severe  ulcerative  infections  of  the 
eye,  in  which  ulcerations  are  especially  localized  on  the  cornea, 
should  search  particularly  for  pneumococci. 

Typing  from  all  these  various  lesions  should  be  done  if  for  no 
other  reason  in  the  interest  of  statistical  knowledge.  From  the 
statistics  furnished  by  the  Rockefeller  Hospital  studies,  as  well  as 
additional  information  which  the  past  five  years  have  furnished 
over  all  the  United  States  and  Europe,  it  seems  fair  to  assume  that 


466  PATHOGENIC   MICROORGANISMS 

type  III  is  probably  the  most  dangerous  of  the  organisms,  followed 
closely  by  types  II  and  I,  in  the  order  named,  and  that  type  IV 
organisms  have,  as  a  rule,  a  less  alarming  prognosis. 


ANTIBODY  FORMATION,  IMMUNITY,   AND  SPECIFIC 

THERAPY 

Recovery  from  a  spontaneous  pneumococcus  infection  confers 
immunity  for  only  a  short  period.  Two  and  three  attacks  of  lobar 
pneumonia  in  the  same  individual  are  not  unusual,  and  it  is  uncer- 
tain whether  even  a  temporary  immunity  is  acquired  in  such  infec- 
tions. Our  recent  knowledge  of  types  has  made  it  seem  not 
impossible  that  successive  attacks  of  pneumonia  may  be  due  to 
consecutive  infection  with  different  types  of  organisms,  thus  leaving 
open  the  possibility  of  the  acquisition  of  prolonged  immunity.  But 
this  seems  doubtful  in  view  of  the  fact  that  Chickering73  and  others 
have  seen  individuals  who  have  had  four  or  five  attacks  within  a 
relatively  short  time.  The  point,  at  any  rate,  is  by  no  means  settled. 
Active  immunization  of  laboratory  animals  may  be  carried  out  by 
various  methods.  The  method  usually  followed  is  to  begin  by  in- 
jecting attenuated74  or  dead  bacteria  or  bacterial  extracts.  Subse- 
quent injections  are  than  made  with  gradually  increasing  doses  of 
living,  virulent  microorganisms.  Great  care  in  increasing  the  dosage 
should  be  exercised  since  the  loss  of  an  animal  after  two  or  three 
weeks'  treatment  by  a  carelessly  high  dose  of  pneumococci  is  not 
unusual.  Wadsworth  centrifugalizes  freshly  grown  pneumococcus 
cultures  and  to  the  pneumococcic  sediment  adds  a  definite  quantity 
of  concentrated  salt  solution.  At  the  end  of  12  hours,  the  pneumo- 
cocci are  dead  and  considerable  destruction  of  the  cell-bodies  has 
taken  place.  Dilution  with  water  until  the  solution  equals  0.85  per 
cent  NaCl  now  prepares  the  emulsion  for  inoculation.  The  sera  of 
animals  immunized  with  pneumococci  contain  active  bactericidal 
substances. 

Specific  agglutinins  in  pneumococcus  immune  sera  were  first 
thoroughly  studied  by  Neufeld75  and  since  then  have  been  made  the 

73  Clnickering — Discussion   on  pneumonia — N.  Y.   Academy  of  Medicine,  April, 
1920. 

74  Eadziewsky,  Zeit.  f.  Hyg.,  xxxvii,  1901;  Neufeld,  Zeit.  f.  Hyg.,  xi,  1902. 
78  Neufeld,  loc.  cit. 


PASSIVE   IMMUNIZATION  467 

subject  of  extensive  studies  by  Wadsworth,70  Hiss,77  and  many  others. 
For  the  sake  of  obtaining  plentiful  growth  for  agglutination  purposes, 
Hiss  has  recommended  cultivation  in  1  per  cent  glucose  broth  with  the 
addition  of  small  amounts  of  sterile  calcium  carbonate  to  absorb  acid 
formed  from  the  glucose.  Pneumococci  do  not  regularly  agglutinate 
in  diluted  immune  sera  and  agglutinations  are  best  studied  in  sus- 
pensions of  more  concentrated  immune  serum.  Agglutination  begins 
at  the  end  of  about  15  minutes,  and  can  be  studied  both  by  formation 
of  clumps  and  by  the  sediment.  See  preceding  paragraphs  on  typing. 
Specific  precipitating  antibodies  have  been  demonstrated  in  pneu- 
mococcus  immune  serum  by  Neufeld,78  Wadsworth,79  Hiss,80  and 
others,  the  organism  for  such  tests  being  brought  into  solution  either 
with  bile  or  with  concentrated  salt  solution.  Such  sera  also  contain 
powerful  opsonic  substances,  or,  as  Neufeld  and  Rimpau81  prefer  to 
call  them,  "bacteriotropins. "  It  seems  most  likely  that  such  phago- 
cytosis-aiding substances  are  most  powerfully  concerned  in  protection 
and  cure.  Clough82  has  reported  an  increased  of  opsonins  at  the  time 
and  crisis,  and  Dochez83  has  shown  that  protective  substances  may 
appear  in  the  serum  at  or  soon  after  the  time  of  crisis.  The  outcome 
of  a  case  according  to  Cole  depends  very  largely  on  the  virulence  of 
the  organism  and  on  the  ability  of  the  body  first  to  limit  the  local 
infection  and  to  prevent  the  invasion  of  the  blood  with  the  organisms. 
In  this  process,  of  course,  the  protective  and  opsonic  bacteriotropic 
substances  would  play  a  most  important  part. 


PASSIVE  IMMUNIZATION  AND  USE  OF  PROTECTIVE  SERA 

The  history  of  attempts  to  produce  sera  for  passive  immunization 
in  man  is  extensive.  Washburn,84  Mennes,85  Pane86  and  many  others 
in  the  past  have  succeeded  in  protecting  animals  with  such  sera,  but 

76  Wadsworth,  loc  cit. 

n  Hiss,  Jour.  Exp.  Med.,  vii,  1905. 

78  Neufeld,  Zeit.  f .  Hyg.,  1902,  xi. 

79  Wadsworth,  loc.  cit. 

80  Hiss,  Jour.  Exp.  Med.,  vii,  1905. 

81  Neufeld  and  Eimpau,  Deut.  med.  Woch.,  1904. 

82  Clough,  Johns  Hopkins  Hosp.  Bull.,  Oct.,  1913. 

83  Dochez,  Jour.  Exp.  Med.,  1913. 

84  Washburn,  Brit.  Med.  Jour.,  1897. 

85  Mennes,  Zeit.  f .  Hyg.,  1897. 
MPane,  Eif.  med.,  1897. 


468  PATHOGENIC   MICROORGANISMS 

with  irregular  results.  The  rational  beginning  based  on  the  recog- 
nition of  different  pneumococcus  types  was  made  by  Neufeld  and 
Haendel  in  Germany,  and  carried  to  a  considerable  degree  of  success 
by  Cole  and  his  associates  at  the  Rockefeller  Hospital  in  New  York. 
By  the  immunization  of  horses  with  the  various  types  of  pneumococci 
mentioned  above,  considerable  success  has  attended  the  use  of  sera 
produced  with  Type  I,  and  less  success  but  great  promise,  that  of  sera 
produced  with  Type  II.  The  injection  of  considerable  quantities  of 
the  homologous  sera  intravenously  at  least  aids  in  sterilizing  the  blood 
stream,  and  upon  this  the  eventual  outcome  of  many  cases  may  depend. 

The  actual  method  of  producing  serum  at  the  present  time  depends 
upon  the  injection  of  horses  with,  at  first,  killed  cultures  and  then 
living  pneumococci.  Horses  are  used  as  in  other  serum  production, 
and  all  the  preliminary  precautions  against  glanders  and  tetanus 
taken.  The  pneumococci  used  are,  of  course,  typed,  since,  as  we  shall 
see  below,  type  I  serum  is  the  only  one  that  so  far  has  yielded  at  least 
hopeful  results.  The  organisms  are  grown  on  suitable  broth,  and 
young  cultures  are  injected.  For  the  early  injections  the  organisms 
are  killed  by  heating  from  56°  to  60°.  The  method  of  Cole  is  to  inject 
daily  for  six  days  the  killed  bacteria  thrown  down  from  50  c.c.  of  a 
12-hour  broth  culture.  A  rest  of  a  week  is  then  given,  and  the  serum 
of  the  horse  tested  for  agglutination.  A  second  series  of  dead  culture 
injections  is  then  carried  out,  and  again  an  interval  allowed.  Again  a 
test  is  made,  and  if  agglutination  is  as  high  as  1—200,  and  0.2  c.c. 
protects  a  mouse  against  0.1  c.c.  of  a  virulent  culture,  the  serum  could 
be  used,  but  Cole  states  that  this  is  not  often  the  case.  After  the 
second  series  of  dead  culture  injections,  living  bacteria  are  injected; 
three  injections  containing  bacteria  from  2.5  c.c.  of  the  original  culture 
are  then  given.  The  actual  methods  of  injecting  horses  will  vary  with 
individual  experience  in  different  places,  but  in  all  cases  the  principle 
is  the  old  one  of  first  injecting  the  living  cultures  with  great  care  not 
to  infect  the  horse,  and  bleeding  determined  by  preliminary  test. 

The  horses  are  bled  in  the  usual  way,  and  the  serum  obtained.  The 
serum  is  taken  up,  stored,  and  handled  as  in  the  case  of  other  pro- 
tective sera. 

Recently  a  great  deal  of  very  interesting  work  has  been  done  upon 
the  relative  purification  of  pneumococcus  anti-sera  from  horse  protein 
by  attempts  to  isolate  the  antibodies  from  whole  serum.  Gay  and 
Chickering87  precipitated  dissolved  pneumococcus  antigen  with  anti- 

8T  Gay  and  Chickering,  Jour.  Exper.  Med.,  21,  1915,  389. 


PASSIVE   IMMUNIZATION  469 

serum,  thus  carrying  down  the  antibodies.  They  then  extracted  these 
precipitates  with  weak  sodium  carbonate  at  42°,  and  in  the  super- 
natant fluid  found  protective  antibodies  which  agglutinate  pneumo- 
cocci.  Huntoon  has  recently  perfected  this  method  of  dissociation  of 
antibody  from  its  antigen  in  a  way  that  promises  practical  usefulness. 
He  treats  large  amounts  of  pneumococci  with  an  excess  of  antibody, 
at  3iy2°.  After  throwing  down  the  pneumococci  with  a  centrifuge,  he 
now  washes  them  with  physiological  salt  solution,  at  almost  the  freez- 
ing point  in  order  to  remove  traces  of  serum,  and  then  treats  them  at 
a  temperature  of  about  40°  with  weak  sodium  bicarbonate  solution,  a 
treatment  which,  as  Landsteiner  and  others  have  shown  with  other 
organisms,  dissociates  antibodies  in  large  amounts  from  the  antigen- 
antibody  complex.  These  antibody  solutions,  if  the  volume  of  final 
solvent  is  either  at  or  about  one-fourth  the  original  serum  volume,  is 
protective  in  approximately  the  same  degree  as  the  original  sera,  and 
is  almost  or  perhaps  completely  protein  free.  These  antibody  solutions 
of  Huntoon  are  at  the  present  time  being  used  experimentally  and 
promising  results  have  already  been  obtained.  Their  intravenous 
injection  produces  an  initial  chill,  probably  due  to  the  non-specific 
reaction  caused,  in  our  opinion,  by  traces  of  bacterial  protein  in  the 
solution.  The  cases  which  we  have  seen  reported,  show,  however,  that 
subsequently  a  specific  reaction  seems  to  occur  which  promises  to 
render  them  perhaps  an  improvement  upon  treatment  with  whole 
serum. 

Standardization  of  Pneumococcus  Serum. — After  a  considerable 
amount  of  discussion  as  to  which  of  the  antibody  reactions  should  be 
used  for  pneumococcus  serum  standardization,  it  has  been  generally 
accepted  that  standardization  by  mouse  protection  is  the  most  reliable 
method.  The  pioneer  work  on  such  standardization  was  done  largely 
by  Neufeld.  Recently,  it  has  been  developed  by  various  pneumococcus 
workers,  Wadsworth  and  Kirkbride,88  the  workers  at  the  Rockefeller 
Hospital,  and  a  number  of  the  manufacturers  of  pneumococcus  serum. 
The  standardization  depends  upon  the  amount  of  serum  necessary  to 
protect  a  white  mouse  of  20  grams  weight  against  a  standard  virulent 
culture. 

One  of  the  most  important  points  in  the  standardization  is  to  use 
a  culture  of  very  great  and  accurately  known  virulence.  This  can  be 
produced  by  passage  through  mice.  The  virulence  of  the  organism 

ss  Wadsworth  and  Kirkbride,  Jour.  Exp.  Med.,  25,  1918. 


470 


PATHOGENIC    M1CROOI  GANISMS 


used  should  be  so  great  that  0.000001  c.c.  of  an  18-hour  broth  culture 
will  kill  a  mouse  in  48  hours.  Broth  dilutions  are  then  prepared  in 
such  a.  way  that  0.5  c.c.  of  each  dilution  contains  varying  quantities  of 
the  pneumococcus  culture  ranging  from  0.2  c.c.  to  0.0000001  c.c. 
These  dilutions  should  be  freshly  .made  in  order  that  the  number  of 
organisms  in  the  tube  shall  not  be  materially  changed  by  growth  or 
death  before  the  tests  are  made.  With  each  of  these  dilutions,  then, 
0.2  c.c.  of  the  serum  to  be  tested  is  mixed  in  a  syringe  and  the  mixture 
immediately  injected  intraperitoneally  into  the  abdominal  wall  just 
above  the  groin.  In  some  laboratories  the  amount  of  serum  used  for 
these  standard  tests  is  0.1  instead  of  0.2  c.c.  The  following  is  a  typical 
protocol  taken  from  a  protection  experiment  by  Dochez  and  A  very, 
which  illustrates  the  method: 


67 

(Group  I). 

A 

69  (GROUP  II) 

Culture 

Controls. 

Serum  I, 
0.2  c.c. 

Serum  II, 
0.2  c.c. 

Controls. 

Serum  I, 
0.2  c.c. 

Serum  II, 
0.2  c.c. 

0.1  c.c. 

Dead 

Survived 

Dead 

Dead 

Dead 

17  hrs. 

11 

41  hrs. 

18  hrs. 

18  hrs. 

0.01 

17  hrs. 

25  hrs. 

18  hrs. 

Survived 

0.001 

41  hrs. 

<  i 

41  hrs. 

18  hrs. 

<  < 

0.0001 

41  hrs. 

11 

41  hrs. 

Dead 

i  < 

18  hrs. 

0.00001 

96  hrs. 

'  ' 

41  hrs. 

18  hrs. 

1  1 

0.000001 

48  hrs. 

t  i 

72  hrs. 

18  hrs. 

(  ( 

(The   quantities   in 
cultures.) 


representing   dose   of   culture   refer  to     18  hour  broth 


The  rule  laid  down  for  sera  by  Cole  and  his  co-workers  is  that 
only  sera  should  be  employed  which  in  doses  of  0.2  c.c.  protects  against 
doses  of  0.1  c.c.  of  a  culture  of  the  above  description.  Variations  in 
these  standards  are  set  up  in  other  laboratories,  and  constant  changes 
are  taking  place  in  this  phase  of  the  work,  but  the  above  will  suf- 
ficiently illustrate  the  principle  applied. 

Methods  of  Serum  Treatment  and  Results. — Hope  of  success  with 
serum  treatment  depends  upon  early  diagnosis  and  immediate  unde- 
layed  typing  of  the  organism,  since,  as  we  shall  see,  the  type  I  serum 
alone  is  at  the  present  time  thought  by  everyone  to  exert  definite 
beneficial  action. 


PASSIVE   IMMUNIZATION  471 

Since  the  serum  is  given  intravenously  it  is  important  to  be  very 
careful  in  estimating  whether  or  not  the  patient  is  horse  serum  sen- 
sitive. Inquiry  as  to  previous  serum  injections  must  be  made,  and 
intradermal  skin  tests  with  horse  serum  arc  done  by  injecting,  intra- 
cutaneously,  with  a  tuberculin  syringe,  a  small  amount,  anywhere 
from  0.05  to  0.02  c.c.  of  a  1-10  horse  serum  dilution.  If  the  injection 
is  properly  made,  in  a  sensitive  subject  within  anywhere  from  five  to 
thirty  minutes  a  large  urticaria-like  wheal  will  arise,  which  will  remain 
for  one-half  to  two  hours,  gradually  disappearing.  In  such  cases  great 
care  should  be  exercised  in  administering  the  serum  and  attempts 
made  at  desensitization  by  the  Besredka  method,  that  is,  gradual 
injection  of  increasing  amounts.  This  is  not  absolutely  reliable,  but 
probably  is  of  great  help  in  most  cases.  It  is  best  to  begin  with  slow 
subcutaneous  injection  of  about  0.02  c.c.  of  horse  serum,  best  diluted 
in  a  total  quantity  of  5  c.c.  of  salt  solution.  This  can  be  repeated, 
gradually  increasing  the  dose  to  one  c.c.  in  three  or  four  instillations 
if  no  untoward  symptoms  appear.  Even  in  these  preliminary  injec- 
tions it  is 'best  to  inject  very  slowly,  and  to  be  sure  that  the  needle  does 
not  enter  a  small  venule,  leaving  about  an  hour  between  injections.  It 
is  difficult  to  lay  down  definite  rules  for  quantity  and  manner  of 
injection,  since  in  each  individual  case  an  experienced  worker  should 
feel  his  way  gradually  in  the  case.  Gradual  desensitization  until  a 
large  intravenous  injection  can  be  given  may  consume  24  hours  or 
more  during  which  time  the  cumulative  doses  may  furnish  a  consider- 
able fraction  of  the  therapeutic  dose. 

When  the  time  for  actual  injection  comes,  the  serum  is  diluted 
with  equal  parts  of  sterile  salt  solution.  This  permits  one  to  inject 
the  substance  more  slowly.  A  special  gravity  apparatus  for  slow 
injection  has  been  devised  for  this  purpose  by  Cole  and  his  co-workers, 
but  with  great  care  the  injection  can  be  done  directly  with  a  large 
syringe.  The  serum  mixture  should,  of  course,  be  brought  to  body 
temperature  before  injection.  The  important  point  is  that  the  injec- 
tion of  the  first  10  c.c.  of  the  serum  should  occupy  at  least  ten  minutes, 
and  this  is  the  critical  time  for  the  development  of  anaphylactic  symp- 
toms during  which  the  patient  must  be  carefully  watched.  Any  signs 
of  respiratory  difficulty,  sudden  changes  in  the  pulse,  etc.,  should  be 
an  indication  for  immediate  cessation  of  the  injection,  which  can  be 
begun  again  when  the  patient  has  returned  to  normal. 

The  total  dosage  advised  by  Cole  and  his  co-workers  is  about  90  to 
100  c.c.  Reinjection  is  a  matter  of  judgment,  and  these  workers  advise 


472  PATHOGENIC   MICROORGANISMS 

that  the  treatment  should  be  vigorously  continued  by  remjections 
every  8  or  10  hours,  as  often  as  it  seems  advisable. 

Serum  disease  which  occasionally  follows  is  similar  to  that  which 
follows  diphtheria  antitoxin  injections. 

In  discussing  the  results  of  serum  treatment  no  final  judgment 
can  be  given.  There  have  been  all  kinds  of  extravagant  claims  both 
as  to  usefulness  and  uselessness  of  the  serum.  Cole's  judgment  we 
consider  to  be  entirely  objective  and  unprejudiced  in  this  matter,  and 
he  states  from  his  own  experience  that  he  has  obtained  no  results  with 
any  serum  except  type  I.  With  this  serum,  however,  he  believes  that 
there  has  been  a  definite  drop  in  the  mortality,  to  about  8  per  cent 
as  contrasted  with  a  normal  mortality  of  20  per  cent,  or  more,  in 
untreated  type  I  cases.  In  general,  this  opinion  seems  to  have  been 
borne  out  by  other  observers  who  have  used  the  method.  It  is  of  the 
greatest  importance  that  in  judging  of  results  of  such  treatment  a 
discrimination  should  be  made  between  cases  that  have  been  treated 
promptly  in  the  early  stages  of  the  disease,  and  those  in  which  the 
treatment  has  been  delayed,  for,  as  every  bacteriologist  knows,  when 
dealing  with  the  pathogenic  Gram-positive  cocci,  that  no  amount  of 
serum  will  save  an  experimental  animal  when  once  an  extensive 
septicemia  has  been  established. 


EPIDEMIOLOGY  OF  PNEUMONIA 

Pneumonia  is  endemic  in  most  well-populated  centers  of  the  world, 
but  seems  to  be  particularly  frequent  in  the  temperate  zones.  The 
disease  is  present  sporadically  at  almost  all  times  of  the  year,  but  is 
particularly  frequent  during  the  colder  months,  usually  reaching  its 
annual  peak  in  this  latitude  during  February  or  March.  It  is  not 
commonly  an  epidemic  disease,  but  may  become  so  under  conditions  of 
crowding,  and  wholesale  exposure  to  wet  and  cold,  incident  to  military 
life,  or  the  life  in  mining  camps,  etc.  Wherever,  in  other  words,  very 
close  association  of  limited  groups  of  people  takes  place  under  condi- 
tions of  poor  hygiene  and  crowding  with  coincident  hardships  of 
various  kinds,  pneumonia  epidemics  are  apt  to  occur.  The  most  exten- 
sive epidemics  which  have  occurred  within  the  last  20  years  are  those 
which  took  place  in  the  South  African  mining  districts,  in  Panama, 
and  during  the  World  War  in  the  camps  and  among  the  armies  at  the 
front.  Pneumonia  of  all  kinds  in  ordinary  times  accounts  for  about 


EPIDEMIOLOGY  OF  PNEUMONIA  473 

10  per  cent  of  the  death  rate,  but  under  conditions  like  those  occurring 
during  the  war,  a  much  larger  percentage  of  all  deaths  are  due  to 
pneumonias.  The  Surgeon  General  estimates  that  in  the  year  1918  the 
total  number  of  deaths  chargeable  to  respiratory  disease  (and  this 
means  with  very  few  exceptions,  death  by  pneumonia  of  one  kind  or 
another)  was  39,701,  out  of  a  strength  of  2,518,499  men,  which 
amounts  to  a  death  rate  of  15.75  per  thousand,  and  82  per  cent  of 
all  deaths  occurring  in  the  Army  during  this  year. 

In  order  to  discuss  the  epidemiological  and  preventive  problem 
concerned  with  pneumonia  with  intelligence,  it  will  be  necessary  to 
discriminate  between  the  so-called  "primary"  pneumonias  and  "sec- 
ondary" pneumonias. 

Inflammations  of  the  lung  may  be  caused  by  a  variety  of  bacteria. 
However,  for  the  purposes  of  considering  the  epidemiology  of  these 
diseases  we  need  take  into  account  only  those  caused  by  various  pneu- 
mococci,  the  hemolytic  streptococci,  and  influenza  bacilli.  The  charac- 
teristics of  an  epidemic  will  vary  considerably  according  to  whether 
the  majority  of  the  cases  are  typical  lobar  pneumonias,  coming  on  with- 
out previous  illness,  or  whether  most  of  the  cases  represent  pulmonary 
infection,  secondary  to  a  preceding  attack  of  influenza  or  to  measles. 
Typical  lobar  pneumonia  is  almost  regularly  a  pneumococcus  infection 
and  this  type  of  the  disease  is  by  far  less  fatal  than  the  other.  The 
secondary  pneumonias  may  be  caused  by  many  different  organisms. 
Even  in  the  same  community,  cases  occurring  at  about  one  and  the 
same  time,  may  be  caused  by  various  pneumococci,  or  streptococci,  the 
majority  of  the  cases  being  due  to  organisms  most  prevalent  in  that 
particular  place.  Such  epidemics  of  secondary  pneumonia  are  the 
types  which  are  most  apt  to  develop  in  times  of  war  or  under  other 
abnormal  community  conditions,  and  this  type  is  far  more  fatal  than 
is  the  typical  lobar  pneumonia. 

Primary  Pneumonias. — That  pneumonia  was  a  communicable  dis- 
ease was  recognized  by  Johannesen  and  other  clinicians  as  early  as  the 
middle  of  the  last  century.  This  point  of  view,  however,  was  not 
generally  accepted  until  quite  recently.  One  of  the  difficulties  that  has 
stood  in  the  way  of  a  more  general  belief  in  the  communicability  of  the 
disease  lias  been  the  fact  that  many  normal  individuals  harbor  in 
mouth  and  throat  pneumococci  which,  until  recent  years,  were  indis- 
tinguishable from  the  organisms  found  in  the  lungs  in  pneumonia.  It 
was  taken  for  granted,  therefore,  that  the  entrance  of  pneumococci  into 


474  PATHOGENIC  MICROORGANISMS 

the  upper  respiratory  passages  could  not,  in  itself,  produce  pneumonia, 
and  that  when  the  disease  occurred,  it  was  in  most  cases  due  to  auto- 
infection,  owing  to  unusual  depression  of  resistance  in  an  individual 
in  whose  mouth  the  pneumococcus  happened  to  be  present.  Moreover, 
there  seemed  to  be  many  instances  of  relationship  between  unusual 
exposure  to  cold  and  wet,  and  the  occurrence  of  pneumonia,  while  it 
was  rarely  possible  to  trace  definitely  the  origin  of  a  case  to  exposure 
to  a  previous  one. 

Recent  recognition  that  there  are  a  number  of  different  types  of 
pneumococci,  investigations  which  were  begun  by  Neufeld  and 
Haendel,89  and  followed  out  more  particularly  in  this  country  by 
Dochez  and  Avery,90  has  furnished  us  with  an  entirely  new  set  of 
facts  for  the  understanding  of  pneumonia  epidemics.  The  types  of 
pneumococci,  as  worked  out  by  these  writers,  have  been  tabulated  in 
another  place.  This  tentative  subdivision  into  types  has  made  it 
possible  to  determine,  in  the  first  place,  whether  or  not  the  ordinary 
mouth  types  are  identical  with  those  found  in  the  lungs  during  pneu- 
monia, and  have  also  permitted  us  to  determine  whether  the  type 
present  in  any  particular  case  was  identical  with  that  found  in  a  pre- 
ceding case  or  in  a  closely  associated  contact.  Following  up  this  trail, 
workers  at  the  Rockefeller  Hospital  have  found  that  over  50  per  cent 
of  the  mouth  organisms  found  in  normal  human  beings  in  and  about 
New  York  city  belong  to  the  heterogeneous  type  IV  group,  whereas, 
over  80  per  cent  of  lobar  pneumonias  are  due  to  types  I,  II,  and  III. 
The  obvious  inference  from  this  reversed  percentage  is  that  lobar 
pneumonia  is  in  most  cases  caused  by  organisms  transmitted  to  the 
victim  from  an  extraneous  source,  and  that  autoinfection  with  the 
patient's  own  mouth  organisms  cannot  be  regarded  as  a  very  common 
occurrence. 

It  should  not  be  concluded  from  this,  however,  that  type  IV  is 
unimportant  as  a  causative  agent  in  the  disease,  since  the  more  recent 
statistics  gathered  during  the  war  show  that  a  considerable  percentage 
of  cases  in  different  localities  may  be  caused  by  this  group.  However, 
since  this  group  is  composed  of  many  apparently  unrelated  members, 
we  can  not  obtain  further  light  upon  the  epidemiological  conditions, 
under  such  circumstances,  at  the  present  time. 

It  has  also  been  shown   bv  Stillmair11  and  others  that  the  more 


"Ncufcld  :IIM|  Ihu'Hdcl,  Arb.  a.  <1.  k.  Gsmlhtsamte,  1010,  34,  293. 

30 Doclicz  and  Avery,  Proc.  Soc.,  Expcr.  Med.  and  Biol.,  14,  1916-37,  126. 

91  Stillman,  Jour.  Exper.  Med.,  26,  1917,  513. 


EPIDEMIOLOGY  OF  PNEUMONIA  475 

virulent  types  I,  II,  and  III,  may  disappear  from  the  mouths  of  con- 
valescents within  three  or  four  weeks,  and  sometimes  sooner,  and  be 
supplanted  at  such  times  by  the  less  virulent  normal  type  IV  strains. 
Stillman  has  also  shown  that  individuals  associated  with  pneumonia 
patients  may  frequently  harbor  organisms  of  the  same  type  as  those 
infecting  the  patients,  and  he  has  found  organisms  corresponding  to 
the  patients'  type  in  the  dust  of  the  sickroom.  It  seems  unquestion- 
able, therefore,  that  there  may  be  carriers  of  virulent  pneumococci 
entirely  analogous  to  the  carrier  states  developed  with  meningococci 
and  other  organisms. 

While  autoinfection,  therefore,  cannot  be  completely  excluded,  it 
seems  probable  that  the  origin  of  most  cases  of  lobar  pneumonia  is  best 
explained  by  the  acquisition  of  a  virulent  pneumococcus  strain,  either 
directly  from  a  case  or  from  a  carrier,  with  a  depression  of  resistance 
due  to  cold,  exposure,  etc.,  coincident  with  the  presence  of  this  virulent 
strain.  In  the  light  of  these  facts  it  is  clear  that  our  sanitary  point  of 
view  in  regard  to  pneumonia  must  be  materially  changed.  We  can 
now  understand  why  localized  epidemics  have  been  so  often  observed 
in  institutions,  war  hospitals,  and  other  crowded  communities,  and  can 
justly  evaluate  the  importance  of  the  transmission  factor  in  the  spread 
of  this  disease.  In  outlining  sanitary  procedures  for  any  disease  it  is 
of  the  utmost  importance  that  such  a  thorough  understanding  of  the 
relative  importance  of  transmission  and  the  susceptibility  factor 
should  be  acquired.  It  is  never  possible  to  carry  out  all  desirable 
measures  of  prevention  completely,  and  it  is,  therefore,  necessary  to 
know  definitely  upon  which  factors  the  greatest  stress  must  be  laid  in 
planning  the  sanitary  campaign. 

In  all  communicable  diseases  the  two  factors  which  influence  spread 
are,  in  the  first  place,  the  transmission  of  the  virulent  organisms,  and, 
in  the  second  place,  the  susceptibility  of  the  recipient.  When  trans- 
mission becomes  general  and  community  susceptibility  is  normally 
high,  as  in  plague,  typhoid,  cholera,  etc.,  epidemics  are  bound  to  spread 
rapidly.  There  are  diseases  like  those  mentioned  above,  as  well  as 
smallpox,  measles,  scarlet  fever,  and  influenza,  in  which  the  suscepti- 
bility of  the  normal,  previously  unexposed  individual  is  so  great  that 
hardly  anyone  sufficiently  exposed,  will  escape.  It  is  plain  that  in  such 
diseases  sanitary  measures  must  be  aimed  particularly  at  the  preven- 
tion of  transmission,  with,  wherever  possible,  artificial  immunization 
of  the  community.  There  are  other  infections,  however,  chief  among 
which  we  believe  is  pneumonia,  in  which  the  resistance  of  normal 


476  PATHOGENIC   MICROORGANISMS 

human  beings  is  comparatively  high.  The  disease  will  not  occur  in  an 
individual  simply  because  he  has  received  the  virulent  organisms  by 
the  proper  route,  from  a  case  or  a  carrier,  but,  in  addition  to  this, 
there  must  be  coincident  hygienic  defects  which  temporarily  depress 
his  resistance.  A  temporary  coincidence  of  two  factors,  therefore, 
transmission  of  the  organisms  and  increased  susceptibility,  must  occur, 
and  it  is  plain  that  in  such  diseases  epidemic  spread  cannot  take  place 
to  any  extensive  degree  unless  both  of  these  factors,  widespread  trans- 
mission and  depression  of  resistance,  become  generalized.  In  such 
cases,  therefore,  while  proper  safeguards  against  dissemination  of  the 
organisms  must  be  developed,  yet  the  efforts  of  the  sanitarian  should 
focus  particularly  upon  measures  by  which  the  normal  resistance  of 
the  community  is  maintained. 

As  a  matter  fact,  pneumonia  epidemics  do  not  occur  as  a  rule  in 
well  nourished  and  housed  communities.  The  epidemic  form  of  the 
primary  disease  develops  only  under  such  conditions  as  those  prevail- 
ing in  army  camps  during  the  cold  weather,  when  men  are  crowded 
together  in  sleeping  quarters,  and  are  developing  colds  and  coughs, 
and  are,  at  the  same  time,  exposed  to  unusual  conditions  of  life,  cold, 
wet,  unaccustomed  food  and  hard  work.  Exceptions  to  this  are,  of 
course,  epidemics  like  those  that  have  occurred  in  Panama  and  South 
Africa,  but  in  these  cases  the  community  in  which  the  disease  was 
prevalent  consisted  very  largely  of  tropical  negroes,  whose  greater 
susceptibility  to  pneumococcus  infection  is  well  known. 

In  a  number  of  epidemics  in  which  we  have  had  the  opportunity 
of  studying  cases,  it  was  quite  apparent  that  the  susceptibility  factor 
was  the  determinative  one  in  individual  instances.  Surveys  showed 
that,  while  it  frequently  happened  that  a  number  of  cases  came  from 
the  same  tent,  the  infections  were  often  of  different  bacterial  types. 
While  direct  transmission  from  one  case  to  another  often  seemed  to 
be  circumstantially  proved,  in  only  a  few  instances  at  a  certain  camp 
in  which  these  studies  were  made,  were  such  cases  associated  with  a 
single  type.  On  the  other  hand,  a  certain  regiment  which  was  ordered 
to  the  shooting  range  during  a  very  wet  spell,  marching  in  the  rain 
and  camping  on  wet  ground,  developed  26  pneumonias  within  16  days. 
Analysis  showed  that  these  cases  were  caused  by  all  four  pneumococcus 
types  without  particular  relationship  between  contacts  and  types.  On 
the  other  hand,  a  considerable  number  of  men  were  found,  in  this 
same  regiment,  at  that  time,  to  be  carriers  of  virulent  pneumococci 
and  streptococci  without  coming  down  with  the  disease. 


EPIDEMIOLOGY  OF  PNEUMONIA  477 

From  evidence  like  this,  we  conclude  that  in  the  sanitation  of 
pneumonia  it  would  be  dangerous  to  lay  too  great  proportionate  stress 
upon  mere  transmission,  but  to  remember  that  the  average  resistance 
to  pneumococcus  infection  of  the  lung  is  fairly  high  among  human 
beings,  and  that  sanitary  precautions  must  include  a  very  rigid  atten- 
tion to  the  factors  of  warmth,  ventilation  in  sleeping  quarters,  ade- 
quate food,  dryiiess  of  feet,  and  avoidance  of  overwork.  In.  communi- 
ties like  those  of  South  Africa,  the  prevention  of  transmission  alone 
cut  short  the  epidemic,  but  we  have  already  pointed  that  the 
susceptibility  factor  was  unusually  high  in  these  communities. 

It  will  rarely  be  necessary  for  sanitarians  working  in  civilized 
communities  to  be  called  upon  to  prevent  epidemics  of  primary  pneu- 
monia. They  will  develop  under  such  conditions  as  those  prevailing 
in  military  camps,  and  which  might  well  be  imagined  as  possible  in 
badly  managed  industrial  communities,  schools,  labor  camps,  etc., 
where  laborers  are  forced  to  sleep  in  ill-ventilated  barracks — are 
crowded  during  working  hours,  or  in  mines,  and  crowded  institutions. 
Such  conditions  may  occur  among  civilian  populations  at  times  of 
famine,  and  penury  incident  to  war.  Primary  pneumonia  epidemics 
will  occur  only  when  crowding,  coincident  with  generalization  of  mild 
respiratory  infections  increases  the  distribution  of  bacteria  and  when, 
at  the  same  time,  the  community  suffers  from  insufficient  shelter  and 
is  perhaps  under-nourished  and  overworked.  The  most  important 
factor  in  the  prevention  of  such  outbreaks,  therefore,  is  attention  to 
the  ventilation  of  sleeping  quarters,  sufficient  number  of  blankets  on 
beds,  dry  feet,  warm  and  plentiful  food,  and  opportunities  for  reason- 
able rest.  If  this  is  combined  with  isolation  of  coughing  and  sneezing 
individuals,  at  least  during  indoor  life,  if  spitting  is  stopped  and  care- 
ful supervision  of  the  cleansing  of  eating  utensils,  sterilization  of 
handkerchiefs,  etc.,  is  enforced,  such  epidemics  should  yield  readily. 

Cases  which  have  been  diagnosed  as  lobar  pneumonia  should  be 
reportable,  like  other  infectious  diseases.  This  has  already  been  intro- 
duced by  a  number  of  health  departments.  In  hospitals  pneumonia 
cases  should  be  treated  as  communicable,  the  cases  isolated,  at  least 
by  maintaining  proper  distance  between  beds,  screening  between  beds, 
and  care  in  the  collection  and  disposition  of  sputum  and  other  secre- 
tions. Care  of  eating  utensils  and  general  cleanliness  should  be  car- 
ried out  with  proper  consideration  of  the  possibilities  of  communica- 
tion that  have  been  spoken  of  above.  In  view  of  the  probability  of 
persistence  of  the  carrier  state  for  four  weeks  or  longer  after  con- 


478  PATHOGENIC   MICROORGANISMS 

valescence,  great  care  in  mouth  disinfection  and  control  of  this  feature 
before  patients  are  returned  to  their  homes  should  be  practiced. 

Secondary  Pneumonias. — In  Secondary  pneumonias  we  are  dealing 
with  an  entirely  different  sanitary  problem.  While  pneumonia  may  be 
secondary  to  a  large  number  of  different  diseases,  the  only  ones  which 
arc  of  distinct  epidemiological  importance  in  this  connection  are 
influenza  and  measles.  There  is  no  epidemic  of  measles  or  influenza  in 
which  there  are  not,  at  the  same  time,  a  considerable  number  of  pneu- 
monias, and  these  pneumonias  are  more  apt  to  take  the  form  of  the 
lobular  or  broncho-pneumonic  type.  In  both  of  these  diseases  there 
is  a  certain  amount  of  inflammation  of  the  bronchial  mucous  mem- 
branes which  seems  to  render  the  patient  particularly  susceptible  to 
secondary  infection  with  virulent  pneumococci  and  streptococci.  The 
peculiar  susceptibility  of  patients  with  measles  and  influenza  to  pneu- 
monia cannot  be  explained  purely  on  the  basis  of  the  mild  bronchial 
inflammation  which  may  be  considered  distinctly  an  integral  part  of 
these  diseases  themselves.  There  is  a  depression  of  resistance  to  pul- 
monary infection  which  is  quite  out  of  proportion  to  that  which  accom- 
panies many  other  conditions  in  which  bronchitis  and  catarrhal 
inflammation  of  the  upper  respiratory  tract  are  common.  Measles 
epidemics  are  fortunately  uncommon  in  urban  communities,  but  may 
assume  dangerous  proportions  in  army  camps,  institutions,  schools, 
etc.  The  mortality  of  uncomplicated  measles  is  low,  but  the  high  mor- 
tality which  so  often  accompanies  epidemics  of  measles  is  almost 
entirely  a  pneumonia  mortality.  In  one  such  epidemic  which  occurred 
at  Camp  "Wheeler  during  the  early  stages  of  our  entrance  into  the 
War,  the  mortality  of  measles  pneumonias  was  29  per  cent.  Sanitary 
measures  under  such  conditions  include,  of  course,  those  aimed  at  the 
prevention  of  the  primary  disease,  as  well  as  attempts  at  preventing 
the  secondary  pneumonias  with  which  we  are  here  particularly  con- 
cerned. But  for  the  saving  of  life,  the  sanitary  attention  to  the  pre- 
vention of  the  secondary  complications  is  by  far  the  more  important 
of  the  two.  With  the  prevention  of  the  primary  disease  we  deal  with 
in  the  chapter  on  Measles,  but  a  few  drops  may  be  said  in  this  place 
concerning  the  important  measures  which  should  be  taken  during 
measles  epidemics  to  prevent  the  occurrence  of  secondary  pneumonias. 
The  principles  of  such  measures  are  twofold,  in  the  first  place  to  pre- 
vent the  case  which  is  coughing  and  spitting  from  transferring  its 
mouth  streptococci  and  pneumococci  to  others.  There  should  be  the 


EPIDEMIOLOGY  OF   PNEUMONIA  479 

most  careful  attention  to  the  cleanliness  of  the  mouth  of  measles 
patients,  both  for  the  reasons  mentioned,  as  well  as  in  order  to  dis- 
courage the  lodgment  of  virulent  organisms  in  the  patient  himself. 
Doctors  and  nurses  should  wear  gauze  masks  when  in  close  contact 
with  the  patient  as  much  for  the  protection  of  the  patient  as  for  their 
own.  A  measles  patient  should  never  be  allowed  to  remain  in  the  same 
ward  with  pneumococcus  or  streptococcus  cases,  and  as  soon  as  a 
measles  case  develops  a  severe  bronchitis  or  pneumonia,  he  should  be 
removed  from  the  measles  ward  into  a  separate  ward  or  room,  since 
he  has  now  become  an  active  danger  to  other  measles  cases.  In  the 
measles  ward  itself  beds  should  be  screened  one  from  the  other,  and 
there  should  be  at  least  five  feet  between  beds.  It  is  of  great  impor- 
tance that  measles  cases  should  be  put  to  bed  and  kept  warm  and  pro- 
tected from  catching  cold  as  soon  as  the  suspicion  of  the  disease  is 
definite,  and  similar  care  should  be  taken  during  the  course  of  con- 
valescence. 

In  the  case  of  influenza  the  conditions  are  similar.  In  the  section 
on  influenza  it  will  be  seen  that  this  disease,  in  its  pure  form,  is  rela- 
tively mild  and  has  a  very  low  mortality.  During  the  second  and 
third  waves  of  an  epidemic,  however,  practically  all  influenza  cases 
show  some  degree  of  respiratory  infection,  and  the  susceptibility  to 
pneumonia  is  so  great  during  this  stage  that  not  only  is  the  percentage 
of  pneumonias  very  high,  but  the  mortality  is  appalling.  During  the 
year  1918  when  the  second  great  influenza  wave  struck  the  American 
Army,  influenza  was  charged,  by  the  Surgeon  General's  Report,  with 
688,869  admissions  among  the  American  troops  for  the  year,  the  dis- 
ease and  its  complications  causing  23,007  deaths.  82  per  cent  of  all  the 
deaths  occurring  in  the  Army  were  due  to  acute  respiratory  disease. 
When  we  consider,  as  we  shall,  in  the  chapter  on  Influenza,  that  all  the 
respiratory  deaths  chargeable  to  influenza  are  really  deaths  from  sec- 
ondary infection,  and  not  to  influenza  itself,  the  appalling  importance, 
and  not  to  influenza  itself,  the  appalling  importance  of  the  secondary 
pneumonias  from  a  sanitary  point  of  view  becomes  apparent.  The  pre- 
ventive measures  that  can  be  taken  in  guarding  against  secondary  pul- 
monary infection  are  chiefly  indirect  ones,  but  are  of  great  importance. 
Among  the  most  important  are  care  of  the  patient  himself.  Studies 
by  Swift,  Harlow  Brooks  and  others  during  the  war  have  shown  that 
immediate  cafe  in  bed,  as  soon  as  the  first  suspicion  of  diagnosis  of 
influenza  is  made,  is  of  the  utmost  value  in  preventing  the  development 
of  secondary  infection.  The  greatest  care  should  be  taken  of  the 


480  PATHOGENIC   MICROORGANISMS 

mouth  of  influenza  patients,  brushing  of  the  teeth  and  cleansing  of  the 
mouth  with  sodium  bicarbonate  solution,  or  salt  solution  gargle  to 
which  20  per  cent  or  30  per  cent  alcohol  can  be  added.  The  patient 
should  be  carefully  guarded  from  infection  by  the  doctors  and  nurses 
who  should  wear  gauze  masks  for  this  purpose.  This  is  not  primarily 
in  our  minds  a  precaution  to  protect  the  physicians  and  nurses,  but 
rather  the  other  way  around.  Dangers  of  transmission  of  pneumo- 
mococci  and  streptococci  from  bed  to  bed  should  be  guarded  against 
as  above  in  the  case  of  measles.  The  pneumonias  which  occur  during 
an  influenza  epidemic  are  very  rarely  due  to  influenza  bacillus  infec- 
tion of  the  lung  alone.  The  fatal  disease  is  caused  by  a  large  variety  of 
organisms,  including  the  various  pneumococci,  the  streptococci  and 
some  others.  In  any  one  particular  place  the  majority  of  cases  may  be 
due  to  one  or  another  of  these  organisms,  this  depending  somewhat 
upon  the  bacteria  which  happen  to  be  most  prevalent  in  this  com- 
munity, and  are  passed  from  mouth  to  mouth  under  conditions  of 
generalized  respiratory  transmission,  occurring  in  this  place.  Thus, 
MacCallum  and  Cole  studied  a  secondary  pneumonia  epidemic  in 
which  hemolytic  streptococci  were  responsible  for  most  of  the  cases, 
but  usually  the  pneumonias  following  in  the  train  of  influenza  are  not 
of  a  single  type,  but  caused  by  any  virulent  member  of  the  lung 
invading  group  of  bacteria  that  happened  by  chance  to  lodge  on  the 
mucous  membranes  of  the  subject  rendered  susceptible  by  his  primary 
disease. 

Thus,  after  an  influenza  epidemic  has  started,  sanitary  measures 
aimed  at  the  prevention  of  the  fatal  secondary  infections  must  focus 
upon  the  transmission  factor  entirely. 

Prophylactic  Vaccination  against  Pneumococcus  Infection. — The 
value  of  prophylactic  vaccination  against  pneumonia  is  not  yet  def- 
inite. Wright92  was  the  pioneer  in  this  work  in  1911,  but  since  he  did 
not  know  about  type  differentiation,  the  value  of  his  work  is  limited. 
The  first  hopeful  experiments  were  made  by  Lister93  in  South  Africa, 
who  typed  his  pneumococci  by  the  usual  agglutination  method,  having 
his  own  types  A,  B,  and  C  (B  and  C  corresponding  respectively  to  II 
and  I  of  our  classification),  made  salt  solution  suspensions  of  the  organ- 
isms, injecting  at  first  six  to  seven  billion,  intravenously.  Later  he 
resorted  to  subcutaneous  inoculations,  giving  three  doses  of  two  billion 


92  Wright  and  Morgan,  Lancet,  1,  1914,  1  and  87. 

93  Lister,  Pub.  South  African  Inst.  f.  Med.  Ees.,  1916,  No.  8. 


EPIDEMIOLOGY  OF  PNEUMONIA  481 

each  at  weekly  intervals.  His  results  were  distinctly  encouraging, 
though  inconclusive.  Cecil  and  Austin94  did  some  experiments  at 
Camp  Upton  in  which  they  vaccinated  12,519  men  against  types  I,  II, 
and  III,  and  their  results  also  were  encouraging,  but  again  uncon- 
vincing. The  problem  is  sufficiently  advanced  to  encourage  wholesale 
use,  if  conditions  exist  under  which  careful  comparison 'with  unvac- 
cinated  controls  can  be  made.  It  cannot,  however,  be  regarded  as 
definitely  established  as  a  prophylactic  measure  in  the  same  way  that 
we  may  regard  typhoid  vaccination.  At  times  of  epidemic  it  should  be 
employed  on  as  large  a  scale  as  possible,  whenever  possible,  since  only 
in  this  way  will  we  be  able  to  judge  of  its  value  eventually.  The 
experiment  can  be  done  without  harm,  since  the  use  of  the  vaccine  is 
contra-indicated  only  in  acutely  sick  individuals,  in  pulmonary  tuber- 
culosis and  nephritis,  in  patients  with  chronic  heart  disease,  and  Cecil 
states  also  in  pregnant  women. 

The  vaccines  which  so  far  have  been  used  consist  of  culture  sus- 
pensions in  salt  solution  killed  by  heating  at  56°  C.,  for  one-half  hour, 
and  standardized  either  by  counting  against  red  blood  cells  or  by 
means  of  the  nephelometer.  0.3  per  cent  tricresol  may  be  added  to 
preserve  the  suspensions  which  are  made  up  so  that  about  one  thou- 
sand million  pneumococci  are  contained  in  1  c.c.  The  dosage  advised 
by  Cecil  is  three  billions  for  the  first  dose,  six  billions  for  the  second 
and  nine  billions  for  the  third. 

Lipo-vaccines  which  consist  of  pneumococcus  suspensions  in  olive 
oil  and  other  vegetable  oil  mixtures  have  been  used,  but  the  method 
of  preparing  these  vaccines  has  not  been  satisfactorily  perfected. 

84  Cecil  and  Austin,  Jour.  Exper.  Med.,  28,  1918,  19. 


CHAPTER   XXIV 

INFLUENZA— THE  DISEASE  AND   ITS  EPIDEMIOLOGY 

THE   INFLUENZA  BACILLUS  AND   OTHER   ORGANISMS  OF   THE 
HEMOPHILIC  GROUP 

The  Disease. — In  no  disease  is  it  so  difficult  to  discuss  the  etio- 
logical  and  epidemiological  problems  as  it  is  in  influenza  since  clinical 
recognition  is  not  sharp  and  is  fraught  with  many  uncertainties.  In 
time  of  epidemics  there  are  certain  characteristics  of  onset,  sequence 
of  symptoms,  and  similarity  of  course  which  make  the  diagnosis  rela- 
tively simple.  But  in  interepidemic  periods  it  is  not  such  an  easy 
matter,  and  the  ordinary  diagnosis  of  grippe  or  influenza  is  at  such 
times  very  largely  a  matter  of  likelihood,  rather  than  actual  recog- 
nition. 

As  will  be  seen,  it  is  not  possible  at  the  present  time  to  base  the 
diagnosis  of  true  influenza  upon  the  isolation  of  the  Pfeiffer  bacillus, 
although  this  had  almost  become  the  habit  until  the  outbreak  of  the 
last  epidemic.  We  cannot  in  this  place  undertake  to  describe  in  detail 
the  clinical  manifestations  of  this  disease.  For  accurate  descriptions 
and  historical  considerations  of  both  the  clinical  and  epidemiological 
problems,  we  may  refer  the  reader  to  Leichtenstern 's  book  published 
in  Nothnagel's  System,  to  Thompson's  Annals  of  Influenza  (London, 
1852)  and  to  the  more  recent  treatises  in  various  medical  text  books.1 
We  wish  to  emphasize,  however,  that  in  our  own  experience  in  the  last 
epidemic,  together  with  a  study  of  the  clinical  records  of  previous  out- 
breaks, we  have  become  convinced  that  pure,  uncomplicated  influenza 
is  a  very  mild  disease  in  which  respiratory  symptoms  may  be  either 
entirely  lacking,  or  may  be  extremely  mild.  This  is  an  important  fact 
to  remember  in  connection  with  etiological  studies,  since  a  very  large 
part  of  the  bacteriological  work  done  on  this  phase  of  influenza  has 
unfortunately  had  to  be  carried  out  on  cases  occurring  late  in  epi- 
demics when  secondary  respiratory  infection  had  become  almost  uni- 
versal. The  pure,  uncomplicated  cases  are  found  in  considerable  num- 
bers only  in  the  early  stages  of  epidemic  outbreaks. 

1  See  also  Zinsser,  "Etiology  and  Epidemiology  of  Influenza,"  in  the  Oxford 
Medical  Papers,  1921. 

482 


INFLUENZA— THE     DISEASE  AND   ITS   EPIDEMIOLOGY        483 

The  characteristics  of  such  cases  are  as  follows :  the  onset  is  almost 
regularly  abrupt.  Typical  cases  become  ill  suddenly,  without  pre- 
monition. More  rarely  there  are  a  few  days  of  general  tired  feeling 
and  malaise,  but  this  is  unusual.  The  first  symptoms  consist  in  head- 
ache, feverishness,  loss  of  appetite,  pains  in  the  back  and  somatic 
muscles,  particularly  in  the  calves  of  the  legs ;  sometimes  suffusion  and 
burning  of  the  eyeballs,  and  often  mild  sore  throat.  The  temperature 
rises  to  anywhere  from  101°  to  104°,  and  this  condition  continues  for 
two  or  three  days,  when  the  patient  gradually  returns  to  normal,  but 
is  left  quite  exhausted.  Very  oc<?asionally,  skin  rashes  appear  in  the 
form  of  erythematous  patches,  not  at  all  uniform  in  appearance  and 
difficult  to  characterize  dermatologically.  The  spleen  is  usually  not 
enlarged.  The  leucocytes  range  from  5,000  to  9,000. 

Such  cases,  if  they  develop  respiratory  complications  at  all,  suffer 
from  nothing  much  more  than  a  mild  laryngitis  or  bronchitis,  which 
appears  toward  the  third  or  fourth  day  and  rapidly  subsides.  The 
first  200  or  300  cases  which  we  saw  during  the  last  epidemic,  among 
American  soldiers  in  France,  developed  practically  no  respiratory 
symptoms  whatever  and  no  focal  lesions  anywhere.  The  disease  was 
so  brief  and  mild  that  it  was  not  recognized  as  influenza  at  first,  and 
was  spoken  of  as  "three-day  fever."  The  morbidity  at  such  times  is 
high,  the  mortality  practically  nil. 

As  we  study  the  literature  of  past  epidemics  and  the  observations 
made  on  the  last  epidemic  in  different  places,  we  find  that  this  experi- 
ence has  been  universal.  In  the  1898  epidemic,  Hey f elder  speaks  of 
Siberian  fever,  which  was  at  first  looked  upon  as  malaria,  and  noted 
the  absence  of  catarrhal  symptoms  in  the  respiratory  organs.  Just 
before  this,  the  epidemic  outbreak  in  Constantinople  was  spoken  of  as 
"dengue  fever,"  and  similar,  unrecognized,  mild  cases  characterized 
the  beginnings  of  the  epidemic  in  Petrograd.  During  the  war-epidemic 
of  1917  and  1918,  Vaughan  and  Palmer  also  noted  the  uncertain  and 
mild  characters  of  the  first  cases  at  Camp  Oglethorpe  (in  March,  1918) 
and  in  Italy,  San  Pietro  suggested  sand-fly  fever  as  a  possible  diag- 
nosis. 

These  considerations  may  seem  irrelevant  in  a  book  of  this  char- 
acter, but  are  important  in  indicating  that  etiological  investigations 
on  influenza  must  include  a  careful  study  of  cases  of  this  kind.  In 
the  later  periods  of  the  first  epidemic  waves,  as  well  as  in  the  second 
and  third  waves,  the  overwhelming  majority  of  the  cases,  because  of 
secondary  infection,  are  respiratory  in  -character,  an  important 


484  PATHOGENIC  MICROORGANISMS 

characteristic  of  the  basic  influenza  being  the  susceptibility  which  it 
creates  to  secondary  respiratory  infection.  The  possibilities  of  error 
in  etiological  investigation  are,  therefore,  obvious. 

Etiology  of  Influenza. — In  1892,  Pfeiffer  described  the  influenza 
bacillus  which  he  isolated  from  the  sputum  of  typical  cases  but  failed 
to  find  in  normal  controls.  The  organisms  were  present  in  large  num- 
bers in  the  sputum  of  early  cases,  and  in  those  with  pulmonary  com- 
plications which  came  to  autopsy;  if  the  bacterial  contents  of  the 
respiratory  passages  were  examined*  from  the  pharynx  progressively 
downward  to  the  lung,  the  influenza  bacilli  were  found  in  increasing 
numbers  as  the  examination  proceeded  toward  the  smaller  bronchi 
and  bronchioles.  Since  that  time,  an  enormous  amount  of  investiga- 
tion has  been  carried  out  in  regard  to  the  relationship  of  the  influenza 
bacillus  to  the  disease,  but  we  cannot  as  yet  state  definitely  whether  or 
not  it  is  the  actual  cause.  During  the  1889  epidemic  and  in  the  years 
following,  much  etiological  research  was  done  and  Huber,  Baiimler, 
Kretz,  Kruse,  Pfuhl  and  many  others  isolated  influenza  bacilli  with 
great  frequency  from  all  the  various  lesions  associated  with  the  disease. 
In  the  interepidemic  periods  influenza  bacilli  were  found  with  less  and 
less  frequency  in  respiratory  infections,  but  still  were  found  to  be 
present  in  a  great  many  individuals  even  when  they  did  not  suffer 
from  clinical  influenza.  It  is  difficult  to  come  to  any  definite  con- 
clusions on  the  etiological  considerations  involved  in  influenza  at  the 
present  time,  and  it  is  quite  impossible  to  detail  the  enormous  amount 
of  work  that  has  been  done  since  1889  on  this  problem.  There  are  few 
organisms  that  appear  in  the  respiratory  tract  that  have  not  been 
thought  of  by  someone  as  possibly  causing  influenza,  but  most  observers 
have  confined  themselves  either  to  the  confirmation  or  refutation  of 
Pfeiffer 's  claim  concerning  the  specific  significance  of  the  small  Gram- 
negative  bacillus  which  he  discovered.  We  feel  confident  that  if  the 
disease  is  a  bacterial  disease  at  all,  no  other  bacteria  need  be  seriously 
considered. 

It  seems  best  to  us  to  summarize  as  briefly  as  possible  the  evidence 
bearing  on  the  etiological  problem  in  influenza,  referring  the  reader 
for  details  to  the  books  mentioned  to  the  numerous  articles  that  have 
appeared  on  the  subject  in  the  last  three  or  four  years,  and  to  our  own 
summary  mentioned  above,  in  the  Oxford  Series. 

In  the  first  place,  let  us  reiterate  that  correct  judgment  of  etio- 
logical work  in  this  disease  must  be  based  upon  the  recognition  that 


INFLUENZA— THE  DISEASE  AND  ITS  EPIDEMIOLOGY       485 

influenza,  in  its  pure  and  uncomplicated  form,  consists  of  the  mild, 
systemic,  febrile  disease  outlined  above.  It  is  the  causation  of  this 
basic  condition  which  constitutes  the  true  etiological  problem.  The 
point  is  to  decide  whether  the  influenza  bacillus  initiates  this  condition, 
or  whether,  like  pneumococci,  streptococci,  and  other  bacteria,  it  may 
not  be  a  secondary  invader,  preliminary  to  which  there  may  have  been 
infection  by  some  other  agent,  perhaps  a  filtrable  virus,  which  opened 
the  path  for  the  secondary  invasion. 

In  favor  of  regarding  the  influenza  bacilli  as  the  primary  cause 
are :  The  frequent  isolation  of  bacilli  from  the  throats  of  the  earliest 
cases;  the  frequency  with  which  these  organisms  have  been  isolated 
from  all  varieties  of  early  and  late  complications ;  the  distribution  of 
the  influenza  bacilli  in  the  bronchial  trees  in  fatal  cases,  and  their 
discovery  in  many  cases  in  pure  culture  at  autopsy ;  the  wide  distribu- 
tion of  the  organisms  throughout  communities  at  times  of  epidemic, 
and  their  gradually  diminishing  frequency  in  normal  and  diseased 
respiratory  passages  as  epidemics  fade  into  the  past. 

The  recent  discovery  by  Parker  in  our  laboratory  of  the  powerful 
poisons  which  can  be  obtained  from  young  influenza  cultures,  has 
further  given  strength  to  the  possibility  that  the  profound  prostration 
and  systemic  symptoms  in  an  influenza  patient  might  be  due  to  absorp- 
tion of  such  poison  from  even  a  small  focus  in  the  throat. 

Recently,  also,  Cecil  and  Blake  carried  out  a  series  of  significant 
experiments  in  which  they  increased  the  virulence  of  influenza  bacilli 
by  passing  them  through  mice  and  inoculated  these  cultures  into 
Philippine  monkeys  by  swabbing  them  into  the  nasal  mucosa  and 
injecting  them  into  the  trachea.  They  obtained  acute  respiratory  dis- 
ease, with  prostration  and  subsequent  respiratory  symptoms,  bron- 
chitis, etc.,  in  these  animals.  The  experiments  are  not  conclusive,  since, 
of  course,  it  was  impossible  to  say  that  the  disease  which  they  pro- 
duced was  analogous  to  influenza  in  the  human  being.  Cecil  also  has 
carried  out  similar  experiments  on  human  volunteers.  The  idea  under- 
lying the  experiments  was  the  assumption  that  the  virulence  of  the 
strains  used  must  be  raised  to  a  certain  pitch  before  simple  introduc- 
tion into  the  nose  can  lead  to  typical  disease.  In  his  human  experi- 
ments, Cecil  obtained  moderate,  local  and  systemic  symptoms  which 
suggested  very  mild  influenzal  attacks.  None  developed  temperature. 
Other  investigators  who  paid  less  attention  to  the  virulence  of  the 
strains,  particularly  Wahl,  White  and  Lyall,  Bloomfield,  Rosenow  and 
McCoy  and  others,  have  obtained  entirely  negative  results. 


486  PATHOGENIC   MICROORGANISMS 

Against  the  assumption  of  the  etiological  importance  of  the  influ- 
enza bacillus,  are  the  occasional  failures  to  find  these  organisms  in 
early  cases,  the  frequent  presence  of  the  bacilli  in  the  throats  of 
normal  individuals  suffering  from  no  typical  influenza,  and  their 
presence  in  interepidemic  periods  in  many  pathological  conditions 
observed  in  clinical  influenza ;  their  frequent  presence  as  complicating 
invaders  in  whooping  cough,  measles,  etc.,  and  the  antigenic  multi- 
plicity of  strains  isolated  at  times  of  epidemic.  The  absence  of  the 
organisms  in  most  early  blood  cultures,  also,  should  be  added  to  the 
negative  evidence. 

Investigations  of  antibodies  in  the  serum  of  cases  that  are  sick 
permit  of  no  etiologic  conclusions,  since  secondary  invasion  might  lead 
to  antibody  formation,  whatever  the  primary  cause. 

Protection  experiments  with  vaccines  have  been  absolutely  incon- 
clusive. 

All  of  this  evidence  must  be  considered  in  connection  with  recent 
experiments  upon  the  possibility  of  causation  by  a  filtrable  virus  in 
influenza,  a  problem  upon  which  much  work  has  been  done,  and  which 
we  must,  therefore,  include  in  our  summary  as  follows : 

FILTRABLE  VIRUS  AND  INFLUENZA. — The  idea  that  a  filtrable  virus 
might  be  the  etiological  agent  in  influenza  is  suggested  by  the  char- 
acteristics of  the  pure  uncomplicated  mild  cases  described  above,  the 
extreme  infectiousness  of  the  disease,  and  the  negative  character  or, 
at  least,  the  lack  of  uniformity  of  the  bacteriological  findings  in  early 
cases.  In  consequence,  a  considerable  number  of  workers  have  attacked 
the  problem  from  this  point  of  view.  In  1918,  Nicolle  and  Lebailly  2 
reported  on  studies  made  on  the  filtration  of  influenzal  virus  and  its 
inoculation  into  animals  and  man.  They  filtered  the  blood  and  nasal 
secretions  of  uncomplicated  grippe  cases,  and  instilled  them  into 
conjunctival  sacs  and  nasal  cavities  of  several  monkeys  (Macaccus 
Sihicus)  and  into  a  number  of  human  volunteers.  They  obtained 
symptoms  within  about  six  days  in  several  monkeys,  and  in  two  men, 
but  were  unable  to  carry  the  infection  into  a  second  generation.  They 
concluded  that  the  filtrates  of  secretions  in  influenza  are  virulent,  and 
can  infect  human  beings  and  certain  monkeys  by  nasal  and  con- 
junctival inoculation. 

Very  soon  after  this,  Dujarric  de  la  Riviere  3  filtered  blood  from 

2  Nicolle  and  Lebailly,  Ann.  de  1'Inst.  Past.,  33,  1919,  385. 

3  Dujarric  de  la  Eiviere,  Gompt.  Rend.  Acad.  des  Sciences,  167,  1918,  406. 


INFLUENZA— THE   DISEASE  AND   ITS  EPIDEMIOLOGY        487 

four  influenza  cases  and  injected  it  into  himself.    He  became  definitely 
ill  with  influenza-like  symptoms. 

Lister  and  Taylor  4  in  1919  carried  out  a  series  of  filtration  experi- 
ments which  may  be  summarized  as  tending  to  refute  the  idea  that  the 
influenzal  virus  is  filtrable.  In  the  same  year,  Bradford,  Bashford 
and  Wilson  5  claimed  that  they  had  produced  disease  in  animals  with 
filtrates  of  the  blood,  sputum  and  other  exudates  from  influenza 
patients,  and  had  grown  from  this  a  minute  filter  passing  body.  '  This 
work  was  later  withdrawn  by  the  writers  themselves.  Claims  of  the 
filtrability  of  the  influenzal  virus  on  the  basis  of  experiments  carried 
out  by  filtration  of  influenzal  secretions  and  inoculation  into  monkeys 
and  man  were  also  made  in  1919  by  Leschke,0  Fejes,7  and  others.  A 
reivew  of  most  of  the  German  etiological  work  is  to  be  found  in  the 
Centralblatt  fur  Bakteriologie,  (Ref.,  68,  1919,  p.  401).  Selter,8  fail- 
ing to  find  influenza  bacilli  with  any  regularity  in  supposedly  typical 
cases,  filtered  the  nasopharyngeal  mucus  and  gargle  water  of  patients  in 
the  early  stages  of  the  disease,  and  sprayed  it  into  his  own  throat  and 
that  of  a  woman  assistant,  both  of  them  inhaling  the  spray.  In  both 
of  them,  after  17  to  20  hours,  he  claims,  mild  influenza  resulted. 
Yamanouchi,  Sakami  and  Iwashima 9  carried  out  more  extensive 
experiments  in  1919.  If  one  could  accept  these  experiments  without 
question,  the  filtrability  of  the  virus  of  influenza  would  be  an  estab- 
lished fact.  In  their  first  experiment  they  emulsified  the  sputum  of 
43  influenza  patients  in  Ringer's  solution.  Part  of  this  they  filtered. 
The  unfiltered  emulsion  they  injected  into  the  noses  and  throats  of  12 
healthy  people.  The  filtrate  of  the  same  emulsion  was  similarly 
injected  into  the  noses  and  throats  of  12  other  healthy  people,  6  of 
whom  had  had  influenza.  All  of  the  24,  except  the  ones  who  had  had 
influenza  recently,  came  down  with  an  influenza-like  malady  after  an 
incubation  of  two  or  three  days.  Following  this,  they  injected  the 
filtrate  of  blood  of  influenza  patients  into  the  noses  and  throats  of  6 
more  healthy  people,  with  similar  positive  results.  Filtrates  of  sputum 
were  inoculated  into  4  healthy  people,  and  4  others  received  filtrates 
of  blood  of  influenza  patients,  subcutaneously.  All  exc.ept  one,  who 


4  Lister  and  Taylor,  Pub.  South  Afric.  Inst.  Med.  Res.,  April  30th,  1919,  No.  12. 

5  Bradford,  Bashford  and  Wilson,  Lancet,  1,  1919,  169. 
"  7,r.sr///,r,    I'.rrl.   klin.  Woch.,    1 1,   191!). 

7  Fejes,  Dent.  nied.  Woch.,  1919,  653. 

8  Selter,   Dout.  mod.  Woch.,   1018,  932. 

8  Yamaiwuchi,  Sakami  and  Iwashima,  Lancet,  1,  1919,  971. 


488  PATHOGENIC  MICROORGANISMS 

had  had  influenza,  came  down  after  two  or  three  days.  Finally,  pure 
cultures  of  Pfeiffer,  and  mixed  cultures  of  Pfeiffer  bacilli  with  pneu- 
mococci,  staphylococci  and  streptococci  were  injected  into  the  noses 
and  throats  of  14  healthy  people  who  had  not  had  influenza.  No 
symptoms  followed  these  injections.  More  recently,  Olitsky  and 
Gates  10  reported  upon  experiments  they  had  made  in  the  18  months 
previous  to  publication,  beginning  during  the  epidemic  wave  of  1918. 
They  intratracheally  inoculated  secretions  from  influenza  patients  in 
the  early  stages,  into  rabbits.  After  a  short  period  these  rabbits 
developed  fever,  leucopenia,  minute  pulmonary  hemorrhage,  and 
pulmonary  edema  and  emphysema.  The  rabbits  did  not  ordinarily 
die  of  the  disease,  but  when  they  killed  them  in  the  active  stages  and 
filtered  the  material  from  these  pulmonary  lesions,  they  were  able  to 
continue  producing  such  disease  in  rabbits  with  the  filtrate.  They 
proved  by  careful  experimentation  that  the  symptoms  and  lesions  they 
obtained  were  not  due  to  bacteria  in  the  ordinary  sense  of  the  word. 
It  is  impossible  at  the  present  time  to  comment  conclusively  on  these 
results,  but  the  evidence  is  submitted  to  show  that  there  is  a  consider- 
able amount  of  evidence  at  the  present  time  which  should  make  one 
conservative  in  definitely  claiming  etiological  relationship  for  the 
influenza  bacillus. 

EPIDEMIOLOGY  OF  INFLUENZA 

As  stated  before,  the  etiological  and  diagnostic  difficulties  in  con- 
nection with  influenza  are  such  that  records  of  the  disease  are  less 
apt  to  be  reliable  than  would  be  similar  records  of  smallpox,  diph- 
theria, etc.  However,  a  great  deal  is  known  about  past  epidemics 
which  have  been  described  with  sufficient  accuracy  to  permit  us  to 
recognize  them  definitely  as  true  influenza  epidemics.  A  great  deal 
has  been  written  in  the  past  and  is  available  in  the  works  of  Caienus 
of  Grief swald  (1579),  Jacques  Pons  of  Lyon  (1596),  Sydenham 
(1675),  Slevogt  (Jean,  1712),  Haygrath  and  Hamilton  (1775), 
Pringle  and1  Huxham,  Massin  (Strassburg,  1858)  and  many  others. 
Leichtenstern,  who  has  written  a  vary  thorough  treatise  on  influenza 
tabulates  the  great  influenza  epidemics  of  the  world  as  follows: 

Less  extensive  outbreaks  seem  to  have  prevailed  in  different  parts 
of  the  world  between  1709  and  1912. 


Olitsky  and  Gates,  Jour.  Exper.  Med.,  33,  1921,  No.  2  and  3. 


EPIDEMIOLOGY  OF  INFLUENZA  480 

Between  1729  and  1733  the  disease,  traveling  from  Russia  west- 
ward, spread  over  Europe  in  two  great  waves,  one  in  1729  and  the 
other  in  1732. 

Another  epidemic  started  on  the  shores  of  the  Baltic  in  1742. 

In  1757— 1758 1  , 

-.^o    Epidemics  occurred  ot  which  we  have  but  poor 
17ol — -Ubz  \  ,  .     , 

geographical  records. 
1767  J 

Of  the  epidemic  of  1742,  Friedrich  states  that  all  but  about  one- 
tenth  of  the  entire  population  of  Germany  was  attacked. 

From  1781  to  1782  an  epidemic  supposed  to  have  started  in  China 
spread  through  Siberia  to  Russia  and  thence  to  Europe. 

Another  traveled  approximately  the  same  route  in  1788. 

The  same  thing  occurred  between  1799  and  1803. 

In  1827  there  was  an  outbreak  in  Europe  less  extensive  than 
most  of  the  others. 

Between  Iy330  anci  ig33  there  were  two  or  three  pandemic  waves, 
the  first  one  supposedly  originating  in  China. 

Other  outbreaks,  again  traveling  from  East  to  West,  occurred 
in  1836  and  1847.  During  this  epidemic  the  Prussian  Army  is  said 
by  Friedrich  to  have  been  attacked  in  its  entire  personnel. 

These  brief  data,  which  bring  us  up  to  the  pandemic  of  1889,  are 
condensed  chiefly  from  Leichtenstern.11 

The  characteristics  of  influenza  epidemics  are  summarized  by 
Leichtenstern  in  a  manner  which  can  be  accepted  as  roughly  describ- 
ing the  actual  facts  on  the  basis  of  experience  with  the  last  war- 
epidemic.  1.  The  disease  appears  in  true  pandemic  waves;  2,  it 
travels  with  tremendous  speed  over  the  globe ;  3,  it  is  characterized 
by  sudden  mass  infection;  4,  it  is  rapidly  burnt  out  after  several 
weeks  in  one  locality;  5,  it  is  independent  of  season  or  weather; 
6,  it  begins  at  first  with  an  enormous  morbidity  and  a  relatively 
slight  mortality;  7,  it  is  but  slightly  influenced  by  age,  sex,  or 
occupation. 

The  second  characteristic  which  we  have  mentioned,  namely,  that 
the  disease  seems  to  originate  in  one  particular  part  of  the  world 
and  from  there  spreads  out  (such  foci  having  been  also  described 
as  existing  in  Asia  (Netter),  China  (Pearson),  etc.),  is  at  the  present 
time  somewhat  in  dispute,  Frost12  and  others  believing  that  the 
last  two  epidemics  probably  started  in  several  places  at  once. 

11  Leichtenstern,  Influenza  in  the  19th  Century,  2nd  Edit.,  Sticker,  Leipzig,  1912. 

12  Frost,  U.  S.  Public  Health  Serv.  Eep.,  No.  550,  August  15th,  1919. 


490  PATHOGENIC   MICROORGANISMS 

The  pandemic  of  1889  probably  started  in  the  East  where  it  is 
quite  likely  that  an  outbreak  of  so-called  "dengue  fever"  in  Con- 
stantinople in  1888  formed  one  of  the  earliest  manifestations.  Late 
in  1888  and  in  early  1889,  it  seems  to  have  appeared  synch ronously 
in  Greenland,  in  Russia  and  in  Siberia.  Ileyfeldcr13  saw  cases  in 
Bokhara  in  May,  1889,  and  wrote  of  its  enormous  westward  speed 
of  travel.  In  October  it  reached  Petrograd,  and  in  November 
entered  Germany.  It  swept  westward  through  France,  Austria  and 
Italy,  reaching  Spain  in  early  December,  New  York  and  London  by 
the  middle  of  December,  and  by  this  time  had  also  reached  the 
United  States  from  the  other  side,  having  traveled  eastward  as  well 
as  westward  from  its  origin. 

It  is  quite  certain  at  the  present  time  that  influenza  is  spread  by 
direct  and  indirect  contact.  The  tempestuous  suddenness  with 
which  it  attacked  whole  communities  formerly  gave  rise  to  the 
opinion  that  it  was  spread  by  other  means,  such  as  air,  dust,  etc., 
but  studies  of  the  last  two  epidemics  seem  to  contradict  this.  It 
does  not  travel  more  rapidly  than  human  communication,  as  shown 
during  the  1889  epidemic  by  Parsons,14  Friedrich,15  and  others. 
Communities  that  are  out  of  touch  with  infected  populations  by 
reason  of  lack  of  communication  (islands,  mountain  tops,  etc.), 
usually  remain  uninfected.  Examples  of  this  were  noted  during 
the  1889  epidemic  on  the  Island  of  Borkum,  and  on  the  Senlis 
mountain.  In  large  cities  the  epidemics  burn  themselves  out  within 
a  relatively  short  time,  while  in  country  communities  where  com- 
munication is  slower  and  the  population  is  scattered,  it  travels  more 
slowly  and  lasts  longer.  According  to  the  studies  of  Abbott  of  the 
epidemic  in  Massachusetts  in  January,  1890,  it  was  shown  that  from 
the  4th  of  January  to  the  10th  of  February,  there  were  about 
800,000  cases,  that  is,  about  40  per  cent  of  the  population,  and  the 
disease  had  practically  burnt  itself  out  in  this  short  period.  In 
London,  the  epidemic  appeared  in  December,  attained  a  death  rate 
of  28.1  per  thousand,  during  February,  and  began  to  decline  in 
March.  During  the  last  pandemic  similar  facts  were  observed,  al- 
though the  state  of  war,  necessitating  the  transportation  of  large 
bodies  of  men  from  one  part  of  the  world  to  another,  rendered 
tracing  of  the  correlationship  of  influenza  and  travel  routes  extremely 

"  Hey i 'elder,  Wien.  klin.  Woch.,  1890,  3,  11. 

**  Parsons,  Local  Government  Board  Eeport,  London,  1893. 

25 Friedrich,  Arb.  a.  d.  k.  Gesundh.,  1894,  Bd.  9. 


EPIDEMIOLOGY  OF  INFLUENZA  491 

difficult.  However,  MacNeal10  who  has  written  up  the  progress  of 
the  epidemic  among  American  troops  in  France  cites  a  number,  of 
cases  where  a  true  connection  between  ship  communication  and 
the  outbreak  of  influenza  could  be  observed.  Hospital  outbreaks, 
such  as  the  one  described  by  Foster  and  Cookson/7  and  prison 
outbreaks,  such  as  the  one  described  by  Stanley18  for  the  San  Quen- 
tin  Prison,  very  definitely  prove  the  paramount  importance  of  con- 
tact infection.  Stanley  shows  definitely  that  the  disease  was  brought 
in  by  an  infected  prisoner,  and  that  prisoners  in  contact  with  this 
one  and  with  other  infected  inmates  contracted  the  disease,  while 
those  who  were  isolated  in  other  buildings  or  not  in  particularly 
close  contact  with  others,  were  spared.  As  to  the  suddenness  with 
which  the  disease  attacks  a  great  many  people  in  a  community, 
the  chief  point  which  would  make  it  seem  that  contact  might  be 
entirely  responsible,  this  can  probably  be  explained  by  the  extreme 
infectiousness,  the  large  percentage  of  susceptibles,  and  the  fact 
that  people  in  the  early  stages  of  the  milder  forms  of  influenza  are 
up  and  about  and  are  freely  mingling  with  their  fellows.  An 
epidemic  is  rarely  recognized  in  a  large  community  until  two  weeks 
or  longer  after  the  first  cases  have  appeared.  Parsons  in  his 
studies  of  the  epidemics  in  England  in  1889  and  1890,  calls  attention 
to  the  fact  that  influenza  is  not  more  rapid  in  its  spread  and  epidemic 
onset  than  was  smallpox  in  the  days  before  vaccination.  He  also 
has  found  evidence  that  shows  that  in  localities  where  the  outbreak 
seemed  particularly  explosive,  this  could  often  be  traced  to  meetings 
of  crowds  at  conventions  or  other  organizations  at  times  just  preced- 
ing the  beginnings  of  the  epidemics.  The  explosiveness  of  the  out- 
breaks during  the  last  epidemic  was  evident,  particularly,  in  military 
communities. 

Influenza  epidemics  are  always  followed  by  secondary  and  tertiary 
waves  during  which  the  disease  after  a  definite  lapse  of  time  seems 
to  return  often  in  a  more  dangerous  form.  This  has  been  noted  in 
almost  all  carefully  studied  epidemics. 

After  the  epidemics  of  1729  and  1730,  secondary  waves  occurred 
in  1732  and  1733.  After  the  1780  to  1781  epidemic,  another  series 
of  waves  followed  in  1782.  The  1788  epidemic  was  followed  by 


1GM<K>Xc(,l,  Arch,  of  Inter.  Mod.,  2.°»,  1919,  657. 

17  Foster  and  Cookson,  Lancet,  2,  1918,  585. 

"Ktunley,  U.  S.  Pub.  Health  Serv.  Eep.,  No.  19,  May  9th,  1919. 


492  PATHOGENIC  MICROORGANISMS 

secondary  waves  that  lasted  as  long  as  1800,  and  similar  observations 
were  made  during  the  epidemics  of  1836  and  1837.  The  most  careful 
studies  of  such  waves  were  made  by  Parsons  during  the  1889 
epidemic.  He  divided  this  epidemic  into  the  first  wave  which  began 
in  England  in  the  winter  of  1889  to  1890,  a  second  wave  in  the 
spring  of  1891,  and  a  third  in  the  winter  of  1891  to  1892.  Frost 
has  similarly  studied  the  American  epidemics,  and  has  come  to 
analogous  conclusions.  Brownlee19  has  attempted  to  establish  a  law 
of  periodicity  for  the  intervals  between  pandemics,  and  for  the 
intervals  between  several  waves  of  each  outbreak.  In  general,  his 
studies  seem  to  show  that  there  is  an  approximate  period  of  ten 
years  between  large  epidemics,  and  that  a  period  of  about  thirty- 
three  weeks  intervenes  between  individual  waves.  We  cannot  go 
into  these  purely  statistical  facts  in  the  present  work,  but  refer 
the  reader  to  papers  by  Brownlee,  and  the  more  recent  paper  by 
Raymond  Pearl.20 

Secondary  and  tertiary  waves  have  certain  characteristics  which 
it  is  important  to  note.  In  contrast  to  the  relatively  mild  onset 
of  the  primary  waves,  these  later  waves  are  marked  by  greater 
severity  of  the  cases,  and  almost  universal  secondary  infection.  The 
disease  takes  on  a  much  more  dangerous  respiratory  form.  The 
mortality  becomes  progressively  higher  during  these  waves  than 
during  the  original  outbreak.  Leichtenstern,  also  agrees  that,  in 
the  secondary  and  tertiary  waves,  that  the  morbidity  is  lower  and 
the  mortality  much  higher.  Similar  observations  have  been  made 
by  Wutzdorff21  for  the  1889  epidemic. 

The  secondary  epidemic  waves  do  not  travel  with  the  same  speed 
and  to  the  same  extent  as  do  the  first  waves.  Cases  are  more  scat- 
tered and  the  period  of  prevalence  is  more  prolonged.  These  waves 
never  stop  abruptly,  but  play  out,  in  gradually  diminishing  ripples, 
into  subsequent  years.  Also,  according  to  Leichtenstern,  these 
secondary  and  tertiary  waves  do  not  seem  to  take  their  origins 
from  a  single  place,  but  crop  up  here  and  there  from  many  scattered 
foci.  As  Netter  says,  "they  have  appeared  in  separate,  synchronous 
or  successive  explosions,  without  connection  between  various  reap- 
pearances in  different  places,  as  this  was  possible  during  the  first 
appearances  in  1889." 


19  Krownlce,  Lancet,  2,  1919,  856. 

=°  Pearl,  E.,  U.  S.  Pub.  Health  Serv.  Rep.,  No.  548,  August  8th,  1919, 

21  Wutzdorff,  Arb.  a.  d.  k.  Gesundh.,  9,  1894,  414, 


EPIDEMIOLOGY  OF  INFLUENZA  433 

AVhat  is  at  the  bottom  of  this  succession  of  waves,  it  is  hard 
to  say.  The  most  natural  explanation  would  be  that  there  is  a 
short  lived  immunity,  conferred  by  the  original  attack,  and  that  the 
second  and  third  waves  appear  at  times  when  the  infectious  agent 
is  still  widely  distributed  while  the  immunity  has  waned.  It  is 
very  difficult  to  get  at  the  facts,  but  serious  attempts  are  being 
made,  particularly  by  Frost  and  others.  Studies  by  Jordan  and 
Sharp22  at  the  Great  Lakes  Training  Station  indicate  that  no  marked 
immunity  existed  twelve  to  fifteen  months  after  the  first  attack. 
This,  too,  seems  to  be  the  conclusion  reached  by  Frost  who  states 
that  in  Baltimore  those  persons  who  were  attacked  during  the  1918 
to  1919  epidemic,  showed  no  relative  immunity  during  the  epidemic 
of  1920.  It  would  seem  in  general  that  an  almost  universal  infection 
of  a  community  with  the  first  mild  disease  conferred  a  short  lived 
immunity.  As  a  consequence  of  this,  the  epidemic  would  burn  itself 
out  and  wane.  Gradual  return  of  susceptibility  in  the  course  of  the 
subsequent  period  of  months,  not,  however,  bringing  the  community 
back  to  the  very  low  universal  resistance  which  it  was  characterized 
by  before,  would  now  create  a  soil  in  which  reintroduction  of  the 
infectious  agent  could  produce  many  cases,  but  in  which  spread 
would  be  less  rapid  and  extensive.  Such  a  view,  however,  must 
be  regarded  as  purely  tentative,  and  it  is  hoped  that  a  final  study 
of  the  statistics  gathered  during  the  great  war  epidemic  will  clear 
this  matter  up. 

It  is  a  difficult  question  to  decide  where  the  last  war  epidemic 
began.  After  the  1889  epidemic,  it  seems  that  the  disease  may  have 
remained  endemic  in  a  great  many  different  parts  of  the  world.  It 
may  have  been  so  universally  distributed  that  we  cannot  speak, 
in  this  case,  of  a  definite  focus  in  China  or  Turkestan,  as  this  was 
done  in  past  epidemics.  Before  1889,  the  world  was  not  so  con- 
tinuously traveled  over  by  large  numbers  of  people.  There  was 
less  travel  by  railroad  communication,  steam-ship  lines,  etc.  It  may 
be  that  this  development  of  civilization  has  completely  altered  the 
epidemic  conditions  prevailing  in  regard  to  influenza.  It  has  been 
suggested  in  the  case  of  the  last  epidemic  however,  that  it  came 
from  the  East,  as  did  previous  outbreaks.  McNalty  in  an  article  in 
Nelson's  System  states  that  the  disease  was  prevalent  in  March, 
1918,  in  China,  and  that  in  April  cases  appeared  on  a  Japanese  ship 
in  a  Chinese  port.  Frost  who  has  given  particular  attention  to  this 

^-Jordan  and  Sharp,  Jour,  of  Infec.,  Dis.,  May,  1920,  p.  463. 


494  PATHOGENIC   MICROORGANISMS 

question,  on  the  other  hand,  finds  that  the  roots  of  the  epidemic  go 
far  deeper  than  this,  since  his  studies  of  statistics  of  respiratory 
diseases  in  the  United  States  seem  to  show  that  as  early  as  December, 
1915,  and  January,  1916,  there  occurred,  in  New  York  and  Cleveland, 
sudden  and  considerable  rises  in  mortality  from  respiratory  dis- 
eases. In  January,  1916,  influenza  was  reported  in  twenty-two  cities 
of  the  Union.  These  epidemics  were  mild  and  attracted  little  at- 
tention. 

During  the  winter  of  1917,  many  so-called  cases  of  influenza 
occurred  in  Europe  among  French  and  British  troops.  In  the  winter 
of  1917,  similar  cases,  supposedly  influenza,  appeared  in  many  Ameri- 
can camps.  MacNeal  states  that  in  November  and  December,  1917, 
and  January,  1918,  there  were  many  cases  of  so-called  influenza  in 
the  American  Expeditionary  Forces.  The  disease  appeared  without 
question  at  Camp  Oglethorpe  in  March,  1918,  a  month  before  it  was 
recognized  in  any  numbers  in  Europe,  almost  at  the  same  time  at 
which  it  seems  to  have  appeared  in  Spain. 

It  cannot  be  said  with  definifeness  just  where  the  lasi  epidemic 
began,  but  if  we  summarize  the  evidence  available  at  the  present 
time,  it  would  seem  that  Frost  is  probably  right  in  that  it  did  not 
begin  in  a  single  place,  as  previous  epidemics  are  said  to  have  begun, 
but  started  in  a  great  many  different  places  almost  at  the  same 
time. 

Like  other  epidemics  it  appeared  in  successive  waves,  the  first 
wave  probably  beginning  in  1917  in  some  places,  in  the  spring  of 
1918  in  others.  There  was  an  interval,  and  then  in  September  and 
October  of  1918  the  second  wave  had  gathered  its  full  velocity.  This 
was  the  really  fatal  wave  all  over  the  world.  The  mortality  was 
enormous.  Pearl  estimates  that  in  the  United  States  alone,  deaths 
from  influenza  were  not  less  than  550,000  and  this  is  approximately 
five  times  the  number  (111,179)  of  American  soldiers  officially  stated 
to  have  lost  their  lives  from  all  causes  in  the  war.  In  the  Surgeon 
General's  report  it  is  stated  that  influenza,  with  its  complications, 
is  charged  with  688.86  admissions  of  American  and  native  troops 
for  the  year  1918,  and  caused  23,007  deaths,  practically  82  per  cent 
of  all  deaths  in  the  Army  being  attributed  to  respiratory  diseases. 

Morphology  and  Staining.— The  bacillus  of  influenza23  (Pfeiffer 
bacillus)  is  an  extremely  small  organism,  about  0.5  micron  long  by 

23 Pfeiffer,  Deut.  med.  Woch.,  ii,  1892;  Zeit.  f.  Hyg.,  xiii,  1892;  Pfeiffer  und 
Beck,  Deut.  med.  Woch.,  xxi,  1893. 


EPIDEMIOLOGY   OF   INFLUENZA 


495 


0.2  to  0.3  micron  in  width.  They  are  somewhat  irregular  in  length, 
but  show  rounded  ends.  They  rarely  form  chains.  They  are  non- 
motile,  and  do  not  form  spores. 

Influenza  bacilli  stain  less  easily  than  do  most  other  bacteria 
with  the  usual  anilin  dyes,  and  are  best  demonstrated  with  10  per 
cent  aqueous  fuchsin  (5  to  10 
minutes),  or  with  Loeffler's  methy- 
lene-blue  (5  minutes).  They  are 
Gram-negative,  giving  up  the 
anilin-gentian  voilet  stain  upon 
decolorization.  Occasionally  slight 
polar  staining  may  be  noticed. 
Grouping,  especially  in  thin  smears 
of  bronchial  secretion,  is  charac- 
teristic, in  that  the  bacilli  very 
rarely  form  threads  or  chains, 
usually  lying  together  in  thick, 
irregular  clusters  without  definite 
parallelism. 

Isolation  and  Cultivation. — Iso- 
lation of  the  influenza  bacillus  is 
not  easy.  Pfeiffer24  succeeded  in 
growing  the  bacillus  upon  serum- 
agar  plates  upon  which  he  had 
smeared  pus  from  the  bronchial 
secretions  of  patients.  Failure  of  growth  in  attempted  subcultures 
made  upon  agar  and  gelatin,  however,  soon  taught  him  that  the 
success  of  his  first  cultivations  depended  upon  the  ingredients  of 
the  pus  carried  over  from  the  sputum.  Further  experimentation 
then  showed  that  it  was  the  blood,  and  more  particularly  the 
hemoglobin,  in  the  pus  which  had  made  growth  possible  in  the 
first  cultures.  Pfeiffer  made  his  further  cultivations  upon  agar, 
the  surface  of  which  had  been  smeared  with  a  few  drops  of  blood 
taken  sterile  from  the  finger.  Hemoglobin  separated  from  the  red 
blood  cells  was  found  to  be  quite  as  efficient  as  whole  blood.  Whole 
blood  taken  from  the  finger  may  be  either  smeared  over  the  surface 
of  slants  or  plates,  or  mixed  with  the  melted  meat-infusion  agar. 
In  isolating  from  sputum,  only  that  secretion  should  be  used  which 


FIG.  49. — BACILLUS  INFLUENZA  ;  Smear 
from  pure  culture  on  blood  agar. 


Pfeiffer,  loc.  cit. 


496 


PATHOGENIC   MICROORGANISMS 


is  coughed  up  from  the  bronchi  and  is  uncontaminated  by  micro- 
organisms from  the  mouth.  It  may  be  washed  in  sterile  water  or 
bouillon  before  transplantation,  to  remove  the  mouth  flora  adherent 
to  the  outer  surface  of  the  little  clumps  of  pus.  The  blood  of  pigeons 
or  that  of  rabbits  may  be  substituted  for  human  blood. 

For  isolation  of  the  organisms  on  plates  from  sputum  or  other 
sources,  the  best  medium  to  use  is  the  Avery  sodium  oleate  blood 
medium  described  in  the  section  on  technique.  The  sodium  oleate 
in  this  medium  inhibits  a  great  many  of  the  contaminating  organisms 
and  seems  to  favor  the  growth  of  influenza  bacilli. 


FIG.  50. — BACILLUS  INFLUENZA;    Smear  from  sputum.     (After  Heim.) 

In  place  of  this  medium,  blood  agar  plates  can  be  used,  rabbit's 
blood  being,  in  our  opinion,  somewhat  more  favorable  than  other 
varieties  of  blood.  It  is  best  prepared  in  small  lots  by  puncturing 
a  rabbit's  heart  with  a  sterile  needle  and  transferring  the  blood 
directly  to  tubes  of  melted  agar. 

For  further  cultivation,  the  best  medium  is  a  chocolate  agar 
made  as  described  in  the  section  on  media,  in  which  a  5  or  10  per 
cent  rabbit's  blood  agar  with  a  PH  of  7.8  is  heated  to  about  90°  C. 
(but  not  boiled),  and  plates  are  prepared  by  shaking  this  brownish 
discolored  medium,  pouring  it  into  plates  or  tubes  and  letting  it 
cool  before  all  the  blood  has  settled  to  the  bottom.  For  fluid  cul- 
tivation, chocolate  broth  may  be  similarly  prepared  and  filtered 
through  cotton  or  paper  while  hot.  This  makes  a  transparent  straw- 
colored  fluid  on  which  influenza  bacilli  grow  with  great  speed  and 


EPIDEMIOLOGY  OF   INFLUENZA 


497 


luxuriance.  In  the  hands  of  J.  T.  Parker  in  this  laboratory,  this 
method  has  given  excellent  results  and  it  is  by  means  of  such  a 
medium  that  she  has  produced  the  toxic  substances  referred  to 
below.  The  fact  that  this  fluid  medium  does  not  contain  much,  if 
any,  hemoglobin,  but  is  very  rich  in  lipoidal  substances,  makes  us 
believe  that  possibly  the  nutritive  substances  in  blood  necessary  for 
the  growth  of  this  bacillus  may  consist  in  the  lipoidal  contents  of 
the  blood  cells,  rather  than  the  hemoglobin.  This,  too,  is  confirmed 
by  recent  observation  of  Avery.25 

'The  general  view,  however,  is  the  one  whic)?  attributes  to  the 
hemoglobin  in  the  blood  the  nutritive  function. 


FIG.  51. — COLONIES  OF  INFLUENZA  BACILLUS  ON  BLOOD  AGAR.     (After  Heim.) 

A  more  luxurious  growth  of  influenza  bacilli  may  be  obtained 
by  cultivation  in  jars  in  which  about  1/10  the  volume  of  air  has  been 
replaced  by  C02. 

For  preservation  of  laboratory  cultures  of  influenza  bacilli,  the 
best  medium  is  fresh,  defibrinated  rabbit's  blood,  kept  at  room 
temperature  in  the  dark.  In  this  way,  laboratory  strains  after 
several  generations  of  cultivation  outside  the  body  can  be  kept 
alive  for  weeks  and  even  months.  They  will  not  keep  well  either 
in  the  ice-box  or  in  the  incubator. 


Avery,  Proc.  Soe.  Exper.  Biol.  and  Med.,  18,  1921,  6. 


498  PATHOGENIC   MICROORGANISMS 

Other  .substances  which,  added  to  neutral  or  slightly  alkalin 
agar,  have  been  used  for  the  cultivation  of  influenza  bacilli  are  the 
yolk  of  eggs'"0  (not  confirmed)  and  spermatic  fluid.27  None1  of  these, 
however,  are  as  useful  as  the  blood  media.  Symbiosis  with  staphy- 
lococci,-8  too,  has  been  found  to  create  an  environment  favorable 
for  their  development. 

Influenza  bacilli  do  not  grow  at  room  temperature.  Upon  suit- 
able, media  at  37.5°  C.  colonies  appear  at  the  end  of  eighteen  to 
twenty-four  hours,  as  minute,  colorless,  transparent  droplets,  not 
unlike  spots  of  moisture.  These  never  become  confluent.  The  limits 
of  growth  are  reached  in  two  or  three  days.  To  keep  the  cultures 
alive,  tubes  should  be  stored  at  room  temperature  and  transplanta- 
tions done  at  intervals  not  longer  than  four  or  five  days. 

Biology. — The  bacillus  is  aerobic,  growing  in  broth-blood  mix- 
tures only  upon  the  surface,  hardly  at  all  in  agar  stab  cultures,  and 
not  at  all  under  completely  anaerobic  conditions. 

As  it  does  not  form  spores,  it  is  exceedingly  sensitive  to  neat, 
desiccation,  and  disinfectants.  Heating  to  60°  C.  kills  the  bacilli 
in  a  few  minutes.  In  dried  sputum  they  die  within  one  or  two 
hours.  They  are  easily  killed  even  by  the  weaker  antiseptics.  Upon 
culture  media  the  bacilli,  if  not  transplanted,  die  within  a  week  or 
less,  the  time  depending  to  some  extent  upon  the  medium  used. 

Toxin  Formation. — The  opinion  in  former  years  has  been  that 
the  poisonous  substances  produced  by  the  influenza  bacillus  were 
in  the  nature  of  endotoxins  and  a  great  many  observers  noted  toxic 
symptoms  on  the  injection  of  whole  cultures  into  rabbits  and  guinea 
pigs.  There  is  no  question  about  the  fact  that  such  cultures  in 
quantities  of  a  cubic  centimeter  or  more  can  exert  powerful  poison- 
ous action.  In  our  laboratory,  Julia  T.  Parker29  recently  showed 
that  culture  filtrates  of  young  influenza  bacilli  would  kill  rabbits 
in  doses  of  from  1.5  c.c.  upward.  The  best  poisons  are  produced 
by  cultivating  the  organisms  on  broth  of  a  PH  of  7.8,  with  5  to  10 
per  cent  defibrinated  rabbit 's  blood.  They  were  also  produced  actively 
in  the  chocolate  broth  produced  by  the  filtration  of  such  rabbit's 
blood  broth  as  described  above.  The  nature  of  these  poisons  is  un- 
certain. The  symptoms  in  the  rabbits  consist  in  marked  prostration, 

"'Nastjulcoff,  Cent.  f.  Bakt.,  Ref.,  xix,  1896. 

27  Cantani,  Cent.  f.  Bakt.,  xxii,  1897. 

28  Grassberger,  Zeit.  f.  Hyg.,  xxv,  1897. 

29  Parker,  Jour.  A.  M.  A.,  72,  1919,  476. 


EPIDEMIOLOGY  OF  INFLUENZA  499 

flattening  out  on  the  bottoms  of  the  cages,  muscular  weakness  and 
death  in  a  considerable  percentage  of  the  cases  within  two  to  six 
hours.  A  characteristic  feature  is  the  incubation  time  which  is 
regularly  between  forty-five  minutes  and  one  hour  and  one-half. 
These  poisons  have  been  studied  in  parallel  series  with  similarly 
produced  streptococcus  and  typhoid  filtrates  by  Zinsser,  Parker  and 
Kuttner30  and  belong  to  a  class  of  substances  probably  non-specific 
and  non-antigenic,  described  by  us  as  "X"  substances,  the  exact 
nature  of  which  is  at  present  uncertain. 

INFLUENZA  BACILLI  NOT  ASSOCIATED  WITH  EPIDEMIC  INFLUENZA. — 
The  question  of  the  etiological  relationship  of  the  influenza  bacillus 
to  the  epidemic  disease  has  been  sufficiently  discussed  above.  It  is 
an  important  fact  that  the  influenza  bacillus  is  a  common  respiratory 
invader  of  man  in  the  interepidemic  periods,  and  without  any 
apparent  relationship  to  the  typical  epidemic  disease.  Subsequent 
to  the  epidemic  of  1889,  its  wide  distribution  was  established  by  a 
multitude  of  investigations.  Pfeiffer31  noted  its  frequent  presence 
in  tuberculous  processes  in  his  early  studies,  and  this  observation 
was  confirmed  by  many  others.  It  is  especially  frequent  in  bron- 
chiectatic  cavities.  Boggs32  reported  a  number  of  such  cases  in 
which  influenza  bacilli  were  present  symbiotically  in  the  cavity  fluids 
in  cases  that  showed  negligible  symptoms.  A  number  of  observers 
have  isolated  influenza  bacilli  from  the  blood  in  cases  that  had  died 
of  other  conditions.  Jaehle33  obtained  the  bacilli  from  the  heart's 
blood  at  autopsy  from  two  scarlet  fever  cases.  He  also  found  the 
organisms  in  blood  culture  at  autopsy  in  fifteen  cases  of  measles. 
In  one  of  these  cases  the  influenza  bacillus  was  present  when  the 
only  other  influenza  bacillus  lesion  present  was  a  tonsillar  infection. 
He  also  found  the  organisms  in  the  blood  five  times  in  nine  cases 
of  chickenpox,  and  twice  in  twenty-four  cases  of  whooping  cough. 
He  found  them  in  the  respiratory  passages  in  fifteen  cases  of  diph- 
theria. Wynekoop34  has  made  similar  studies,  especially  in  con- 
nection with  lesions  of  the  larynx,  and  in  chronic  laryngitis  he  found 
the  organisms  in  pure  culture.  He  described  a  special  form  of  severe 
tonsillitis  due  to  the  influenza  bacillus.  Some  of  these  were  clinically 

30  Zinsser,  Parker  and  Kuttner,  Jour.  Exper.  Biol.  and  Med.,  18,  1920,  49. 

81  Pfeiffer,  Deut.  med.  Woch.,  18,  1892,  28. 

32  Boggs,  130,  1905,  902. 

83  Jaehle,  Zeit.  f .  Heilkd.,  22,  1901,  190. 

34  Wynelcoop,  Jour.  A.  M.  A.,  40,  1903,  574. 


500  PATHOGENIC   MICROORGANISMS 

mistaken  for  diphtheria.  Madison35  collected  from  the  literature 
thirty  cases  in  which  influenza  bacilli  were  present  in  the  blood 
during  life. 

Influenza  bacilli  in  the  meninges  have  been  described  by  Woll- 
stein36 and  others  and  we  have  seen  them  in  a  number  of  cases  in 
they  were  associated  in  this  location  with  streptococci.  Influenza 
bacillus  meningitis  is  not  uncommon  in  children. 

In  diseases  of  the  cavities  of  the  skull,  the  antrum  of  Highmore, 
the  frontal  sinuses  etc.,  influenza  bacilli  may  be  chronically  present 
and  have  been  held  responsible  for  intermittent  attacks  of  asthma. 

Wollstein  has  made  an  extensive  study  of  the  presence  of  in- 
fluenza bacilli  in  children  at  the  Babies  Hospital  in  New  York.  In 
the  interepidemic  periods,  Wollstein  has  found  them  present  fre- 
quently in  bronchopneumonia,  less  frequently  in  cases  of  lobar  pneu- 
monia. In  thirteen  cases  of  bronchopneumonia  at  autopsy  she  found 
the  organism  three  times.  Six  times  she  found  them  in  connection 
with  tuberculosis.  Other  workers,  as  well  as  Wollstein,  have  fre- 
quently found  the  organisms  in  whooping  cough  where  they  were 
reported  as  being  almost  regularly  present  after  the  disease  was 
well  established.  In  the  lungs  in  measles,  scarlet  fever  and  diph- 
theria their  presence  is  very  frequent.  Wollstein  states  that  she 
has  found  the  organisms  rarely  in  the  throats  of  healthy  infants, 
and  that  whenever  it  was  present,  it  seemed  to  have  a  definitely 
aggravating  influence  upon  the  existing  disease. 

The  carrier  state  may  persist  after  infection  with  influenza  bacilli 
for  long  periods.  During  epidemics  this  seems  to  be  particularly 
important  as  found  by  the  investigations  of  Pritchett  and  Stillman37 
and  of  Opie38  and  his  collaborators. 

Varieties  of  the  Influenza  Bacillus. — A  great  many  investigators 
have  reported  organisms  almost  identical  with  the  influenza  bacillus 
which,  however,  they  have  regarded  as  sufficiently  different  to  be 
considered  as  distinct  types. 

It  is  the  opinion  of  the  workers  at  the  New  York  Department 
of  health,  moreover,  that  the  Gram-negative,  hemophilic  organisms 
found  in  trachoma  must  be  regarded  also  as  belonging  to  the  general 
influenza  bacillus  type,  and  cannot  be  sharply  separated. 


35  Madison,  Amer.  Jour.  Med.  Sc.,  139,  1910,  527. 

36  Wollstein,  Jour.  Exper.  Med.,  1905,  7,  335. 

87  Pritchett  and  Stillman,  Jour.  Exper.  Med.,  29,  1919,  259. 

^Opic,  et  al,  Surgeon  General's  Rep.,  Jour.  A.  M.  A.,  72,  1919,  168. 


EPIDEMIOLOGY  OF  INFLUENZA  501 

The  recent  epidemic  has  given  much  opportunity  for  bac- 
teriological and  serological  study  upon  influenza  bacilli  from  many 
different  types  of  lesion,  from  many  different  parts  of  the  world. 
The  result  of  these  studies  has  been  confusing.  Studies  of  Valentine 
and  Cooper39  have  shown  that  agglutination  reactions  do  not  in- 
dicate hemogenicity  of  the  influenza  group.  In  a  large  number  of 
isolations  and  agglutinin  reactions,  they  found  that  very  few  of 
the  strains  fell  into  antigenically  identical  groups.  Of  ten  autopsy 
strains  isolated  by  them,  all  strains  seemed  to  be  distinct.  Of 
seventy-three  miscellaneous  strains,  no  two  strains  were  identical. 
Two  strains  from  different  individuals  were  found  to  be  identical, 
but  in  a  family  in  which  there  were  six  cases  of  influenza,  all  the 
different  races  were  distinct.  It  would  seem  that  either  influenza 
bacilli  were  composed  of  an  infinite  number  of  antigenic  varieties 
or  that  the  agglutinin  reaction  in  these  particular  organisms,  be- 
cause of  their  small  size,  is  not  a  suitable  test  for  biological  relation- 
ship. For  the  present,  the  biological  subdivision  of  the  influenza 
bacilli  into  well  defined  groups  cannot  be  regarded  as  settled. 

Dr.  Anna  Williams40  has  recently  studied  hemoglobinophilic 
bacilli  isolated  from  the  eye  in  cases  of  trachoma.  She  believes  that 
trachoma  is  probably  caused  by  bacteria  of  this  group.  At  first  an 
acute  infection  or  acute  conjunctivitis  occurs.  Later  when  chronic 
productive  inflammation  supervenes  the  clinical  picture  is  that  of 
trachoma. 

Experimental  infection  of  animals  reveals  susceptibility  only  in 
monkeys.  Pfeiffer  and  Beck41  produced  influenza-like  symptoms  in 
monkeys  by  rubbing  a  pure  culture  of  the  bacillus  upon  the  un- 
broken nasal  mucosa.  Intravenous  inoculation  in  rabbits  produced 
severe  symptoms,  but  the  bacilli  do  not  seem  to  proliferate  in  these 
animals,  the  reaction  probably  being  purely  toxic.  Cultures  killed 
with  chloroform  may  produce  severe  transient  toxic  symptoms  in 
rabbits.42  Immunity  produced  by  an  attack  of  influenza,  if  present 
at  all,  is  of  very  short  duration. 

PSEUDO-INFLUENZA   BACILLUS. — In   the   broncho-pneumonic   proc- 


39  Valentine  and  Cooper,  Rep.  84,  Dept.  Health,  City  of  N.  Y.,  1919,  December. 
""/>/•.  Anna   IVUHdnix,   Ir.lcr.  Congress  of  Hygiene  and  Demography,  Washing- 
ton,   1912. 

41  Pfeiffer  mid  Beck,  Deut.  mod.  Woeh.,  xxi,  1893. 

42  Pfeiffer,  loc  cit. 


502  PATHOGENIC   MICROORGANISMS 

esses  of  children,  Pfeiffer43  found  small,  non-motile,  Gram-negative 
bacilli,  which  he  was  forced  to  separate  from  true  influenza  bacilli 
because  of  their  slightly  greater  size,  and  their  tendency  to  form 
threads  and  involution  forms.  These  microorganisms  are  strictly 
aerobic  and  grow,  like  true  influenza  bacilli,  only  upon  blood  media. 
They  are  differentiated  entirely  by  their  morphology  upon  twenty- 
four-hour-old  blood-agar  cultures.  Wollstein/44  who  has  made  a 
careful  study  of  influenza-like  bacilli,  both  culturally  and  by  agglu- 
tination tests,  has  come  to  the  conclusion  that  these  bacilli  are  so 
similar  to  the  true  influenza  organisms  that  the  term  pseudo-influenza 
should  be  discarded.  Strains  of  similar  bacilli  isolated  from  cases  of 


FIG.  52. — KOCH-WEEKS  BACILLUS. 

pertussis,  while  differing  from  the  others  in  some  of  their  character- 
istics, could  not  properly  be  maintained  as  distinct  species. 

KOCH-WEEKS  BACILLUS. — Koch,45  in  1883,  Weeks46  and  Kartulis, 
in  1887,  described  a  small  Gram-negative  bacillus  found  in  connec- 
tion with  a  form  of  acute  conjunctivitis  which  occurs  epidemically. 
The  bacillus  is  morphologically  similar  to  B.  influenzae,  but  is 
generally  longer  than  this  and  more  slender.  The  bacilli  grow  only 
at  incubator  temperature.  Kecent  studies  by  Anna  Williams  at  the 


43  Pfeiffer,  Zeit.  f.  Hyg.,  xiii,  1892. 

14  Wollstein,  Jour.  Exp.  Med.,  viii,  1906. 

45  Koch,  Arb.  a.  d.  kais.  Gesundheitsamt,  iii ;  Cent.  f.  Bakt.,  1,  1887. 

46  Weeks,  N.  Y.  Eye  and  Ear  Infirmary  Kep.,  1895;  Arch.  f.  Augenheilk.,  1887. 


EPIDEMIOLOGY  OF  INFLUENZA  503 

New  York  Department  of  Health  seem  to  indicate  that  the  Koch- 
Weeks  bacillus  may  be  merely  a  variety  of  the  true  influenza  bacillus. 

BACILLUS  OF  PLEURO-PNEUMONIA  OF  RABBITS. — This  is  a  small  Gram- 
negative  bacillus,  described  by  Beck,  not  unlike  that  of  influenza.  These 
microorganisms  are  slightly  larger  than  the  Pfeiffer  bacilli  and  grow  upon 
ordinary  media  even  without  animal  sera  or  hemoglobin. 

BACILLUS  MURISEPTICUS  AND  BACILLUS  RHUSIOPATHI^E. — While  mor- 
phologically similar  to  the  microorganisms  of  this  group,  these  bacilli  are 
culturally  easily  separated  because  of  their  luxuriant  growth  on  simple  media. 
The  last  two  microorganisms  are  more  closely  related  to  the  groups  of  the 
bacilli  of  hemcrrhagic  septicemia. 


CHAPTER  XXV 

BOEDET-GENGOU  BACILLUS,  WHOOPING  COUGH  AND  MORAX-AXEN- 
FELD  BACILLUS,  ZUE  NEDDEN'S  BACILLUS,  DUCREY  BACILLUS 

BORDET-GENGOU  BACILLUS 

("Microbe  de  la  Coqueluche,"  Pertussis  bacillus,  Bacillus  of  whooping- 
cough.) 

WHOOPING  cough  is  endemic  in  most  cold  countries  and,  on 
occasion,  in  schools,  infant  asylums  and  other  places  where  children 
are  crowded,  may  assume  epidemic  proportions.  Occasional  cases 
may  occur  in  adults,  though  they  are  rare.  The  disease  seems  to 
occur  sporadically  all  through  the  year  in  large  centers,  but  is 
more  common  during  the  winter.  According  to  Rosenau1  suscep- 
tibility is  pretty  general  and  he  states  that  the  disease  causes  at  least 
10,000  deaths  a  year  in  the  United  States.  Indeed,  Eosenau  who 
has  analyzed  the  statistics  for  the  United  States  for  the  year  1910, 
states  that  whooping  cough  caused  almost  as  many  deaths  as  scarlet 
fever.  It  is  the  pulmonary  complications  that  follow  on  the  initial 
infection,  however,  which,  in  this  disease,  are  responsible  for  the 
deaths  and  it  is  the  subacute  and  chronic  inflammations  of  the  lung 
which  lead  to  prolonged  illness  and  .pave  the  way  for  tuberculosis 
and  other  secondary  infections.  For  sanitary  purposes,  an  arbitrary 
incubation  time  of  about  two  weeks  has  been  regarded  as  probably 
nearest  the  facts.  During  the  incubation  time,  the  infectiousness 
does  not  seem  to  be  great,  the  most  contagious  period  being  the 
first  few  days  of  the  actual  disease  when  the  Bordet-Gengou  bacilli 
are  brought  up  in  large  numbers.  It  is  at  this  time  only  that  pure 
cultures  of  the  organisms  can  be  obtained.  Very  soon  after  the 
onset,  influenza  bacilli  and  other  secondary  invaders  are  found. 
Transmission  is  probably  by  direct  and  indirect  contact  as  in  other 


1  Rosevau,  Preventive  Medicine  and  Hygiene,  D.  Appieton  and  Co.,  New  York, 
1921. 

504 


BORDETrGENGOU  BACILLUS 


505 


respiratory  infections,  and  the  general  infectiousness  is  very  high, 
since  susceptible  children  definitely  exposed  do  not  often  escape. 
After  the  disease  has  set  in,  the  patient  may  remain  infectious  for 
others  all  through  the  disease  and  long  into  convalescence.  It  is 
best  to  keep  children  isolated  for  several  weeks  after  the  cough 
has  subsided.  It  has  been  possible  to  reproduce  conditions  simu- 
lating the  disease  in  monkeys  and  in  dogs,  and  there  is  a  suspicion 
that  the  disease  may  be  transmitted  by  domestic  animals,  especially 
dogs.  Though  this  is  not  absolutely  certain,  Rosenau  and  other 
sanitarians  advise  care  in  this  regard. 

Prevention  consists  in  early  diagnosis  and  isolation,  exclusion 
from  school  and  absolute  avoidance  of  close  contact  with  other 
children.  Quarantine  should  continue,  as  stated  above,  for  several 
weeks  after  the  cough  has  completely  subsided. 

In  1900  Bordet  and  Gengou2  observed  in  the  sputum  of  a  child 
suffering  from  pertussis  a  small  ovoid  bacillus  which,  though  similar 
to  the  influenza  bacillus,  showed  a  number  of  morphological  char- 
acteristics which  led  them  to  regard  it  as  a  distinct  species.  As  they 
were  at  first  unable  to  cultivate  this  organism,  their  discovery 
remained  questionable  until  1906,  when  cultivation  succeeded  and 
the  biology  of  the  microorganism  was  more  fully  elucidated. 

Morphology. — The  morphology  of  this  organism  is  described  by 
them  as  follows:  The  organism  in  the  sputum,  early  in  the  disease, 
is  scattered  in  enormous  numbers  indiscriminately  among  the  pus 


**  — 


FIG.  53. — BORDET-GENGOU  BACILLUS. 

cells,  and  at  times  within  the  cells.  It  is  extremely  small  and  ovoid, 
and  frequently  is  so  short  that  it  resembles  a  micrococcus.  Often 
its  polos  stain  more  deeply  than  the  center.  In  general,  the  form 


-  Bordet  et  Gengou,  Ann.  de  1  'inst.  Pasteur,  1906. 


506  PATHOGENIC   MICROORGANISMS 

of  the  organisms  is  constant,  though  occasionally  slightly  larger 
individuals  are  encountered.  They  are  usually  grouped  separately, 
though  occasionally  in  pairs,  end  to  end. 

Compared  with  the  influenza  bacillus  in  morphology,  the  bacillus 
of  pertussis  is  more  regularly  ovoid  and  somewhat  larger.  It  has, 
furthermore,  less  tendency  to  pleomorphism  and  involution. 

Staining. — The  Bordet-Gengou  bacillus  may  be  stained  with  al- 
kaline methylene-blue,  dilute  carbol-fuchsin,  or  aqueous  fuchsin  solu- 
tions. Bordet  and  Gengou  recommended  as  a  staining-solution 
carbolated  toluidin-blue  made  up  as  follows: 

Toluidin-blue    5  gms. 

Alcohol    100  c.c. 

Water 500  c.c. 

Allow  to  dissolve  and  add  500  c.c.  of  5  per  cent  carbolic  acid  in  water. 
Let  this  stand  one  or  two  days  and  filter. 

Stained  by  the  method  of  Gram,  the  bacillus  of  Bordet  and 
Gengou  is  decolorized. 

Cultivation. — Early  attempts  at  cultivation  made  by  the  discov- 
erers upon  ordinary  ascitic  agar  or  blood  agar  were  unsuccessful. 
They  finally  obtained  successful  cultures  from  sputum  by  the  use 
of  the  following  medium : 

One  hundred  grams  of  sliced  potato  are  put  into  200  e.c.  of  4  per 
cent  glycerin  in  water.  This  is  steamed  in  an  autoclave  and  a 
glycerin  extract  of  potato  obtained.  To  50  c.c.  of  this  extract,  150 
c.c.  of  6-per-cent  salt  solution  and  5  grams  of  agar  are  added.  The 
mixture  is  melted  in  the  autoclave  and  the  fluid  filled  into  test  tubes, 
2  to  3  c.c.  each,  and  sterilized.  To  each  tube,  after  sterilization,  is 
added  an  equal  volume  of  sterile  defibrinated  rabbit  blood  or  prefer- 
ably human  blood,  the  substances  are  mixed,  and  the  tubes  slanted. 

On  such  a  medium,  inoculated  with  sputum,  taken  preferably 
during  the  paroxysms  of  the  first  day  of  the  disease,  colonies  appear, 
which  are  barely  visible  after  twenty-four  hours,3  plainly  visible 
after  forty-eight  hours.  They  are  small,  grayish,  and  rather  thick. 
After  the  first  generation  the  organisms  grow  with  markedly  greater 
luxuriance  and  speed.  On  the  potato-blood  medium  after  several 
generations  of  artificial  cultivation,  they  form  a  grayish  glistening 
layer  which,  after  a  few  days,  becomes  heavy  and  thick,  almost 


3  Wollstein,  Jour.  Exp.  Med.,  xi,  1909. 


BORDET-GENGOU  BACILLUS  507 

resembling  the  growth  of  typhoid  bacilli.  In  these  later  generations, 
also,  they  develop  readily  upon  plain  blood  agar  or  ascitic  agar  and 
in  ascitic  broth  or  broth  to  which  blood  has  been  added.  In  the 
fluid  media  they  form  a  viscid  sediment,  but  no  pellicle. 

Culturally,  the  bacillus  varies  from  B.  influenzae  in  growing  less 
readily  on  hemoglobin  media  than  the  latter,  on  first  cultivation 
from  the  sputum.  Later  it  grows  much  more  heavily  on  such  media 
and  shows  less  dependence  upon  the  presence  of  hemoglobin  than 
does  B.  influenzae.  It  also  grows  rather  more  slowly  than  the  influ- 
enza bacillus.  It  is  strictly  aerobic  and  in  fluid  cultures  is  best 
grown  in  wide  flasks  with  shallow  layers  of  the  medium. 

The  Bordet-Gengou  bacillus  grows  moderately  at  temperatures 
about  37.5°  C.,  but  does  not  cease  to  grow  at  temperatures  as  low 
as  5°  to  10°  C.  On  blood  agar  and  in  ascitic  broth  it  may  remain 
alive  for  as  long  as  two  months  (Wollstein). 

Pathogenicity. — As  regards  the  pathogenicity  and  etiological 
specificity  of  this  organism  for  whooping-cough,  no  positive  state- 
ment can  as  yet  be  made.  The  fact  that  it  has  been  found  in  many 
cases  in  almost  pure  cultures  during  the  early  paroxysms,  renders  it 
likely  that  the  organism  is  the  specific  cause  of  the  disease.  Mallory 
and  Homer  4  have  found  bacilli  appearing  to  be  Bordet  and  Gengou 's 
organisms  lying  between  the  cilia  in  the  tracheal  epithelium  of 
whooping  cough  cases  that  have  come  to  autopsy.  However,  in 
early  cases  true  influenza  bacilli  have  been  often  found,  and  these 
latter  seem  to  remain  in  the  sputum  of  such  patients  for  a  longer 
period  and  in  larger  numbers  than  the  bacillus  of  Bordet  and 
Gengou.  Endotoxins  have  been  obtained  from  the  cultures  of  the 
bacilli  by  Bordet  and  Gengou  by  the  method  of  Besredka.5  The 
growth  from  slant  cultures  is  washed  up  in  a  little  salt  solution,  dried 
in  vacuo,  and  ground  in  a  mortar  with  a  small,  measured  quantity  of 
salt.  Finally,  enough  distilled  water  is  added  to  bring  the  salt  into  a 
solution  of  0.75  per  cent  and  the  mixture  is  centrifugalized  and  de- 
canted. One  to  two  c.c.  of  such  an  extract  will  usually  kill  a  rabbit 
within  twenty-four  hours  after  intravenous  inoculation.  Subcu- 
taneous inoculation  produces  non-suppurating  necrosis  and  ulcera- 
tion  without  marked  constitutional  symptoms. 

Inoculation  of  monkeys  with  the  bacilli  themselves  by  the  respira- 
tory path  has  failed  to  produce  the  disease. 

4  Mallory  and  Homer,  Journ.  Med.  Ees.  xxvii,  1912,  p.  115. 
fi  Bordet,  Bull,  de  la  Soc.  Eoy.  de  Brux.,  1907. 


508  PATHOGENIC  MICROORGANISMS 

Specific  agglutinins  may  be  obtained  in  immunized  animals  which 
prove  absolutely  the  distinctness  of  this  organism  from  Bacillus  influ- 
enzas.6 In  the  serum  of  afflicted  children  the  agglutination  is  too 
irregular  to  be  of  value. 

Specific  complement  fixation  with  the  serum  of  patients  is  reported 
by  Bordet  and  Gengou,  but  failed  in  the  hands  of  Wollstein. 


MORAX-AXENFELD  BACILLUS 

In  1896  Morax 7  described  a  diplo-bacillus,  which  he  associated 
etiologically  with  a  type  of  chronic  conjunctivitis  to  which  he  applied 
the  name  " conjonctivite  subaigue."  Soon  after  this,  a  similar  micro- 
organism was  found  in  cases  corresponding  to  those  of  Morax  by 
Axenfeld.8  The  condition  which  these  microorganisms  characteris- 
tically produce  is  a  catarrhal  conjunctivitis  which  usually  attacks  both 
eyes.  The  inflammation  is  especially  noticeable  in  the  angles  of  the 
eye,  most  severe  at  or  about  the  caruncle.  There  is  rarely  mujch 
swelling  of  the  conjunctiva  and  hardly  ever  ulceration.  The  condition 
runs  a  subacute  or  chronic  course.  Its  diagnosis  is  easily  made  by 
smear  preparations  of  the  pus  which  is  formed  with  especial  abun- 
dance during  the  night. 

Morphology. — In  smear  preparations  from  the  pus,  the  micro- 
organisms appear  as  short,  thick  bacilli,  usually  in  the  form  of  two 
placed  end  to  end,  but  not  infrequently  singly  or  in  short  chains. 
Their  ends  are  distinctly  rounded,  their  centers  slightly  bulging, 
giving  the  bacillus  an  ovoid  form.  They  are  usually  about  two 
micra  in  length. 

They  are  easily  stained  by  the  usual  anilin  dyes,  and,  stained  by 
the  method  of  Gram,  are  completely  decolorized. 

Cultivation. — The  Morax-Axenfeld  bacillus  can  be  cultivated 
only  upon  alkalin  media  containing  blood  or  blood  serum. 

It  grows  poorly,  or  not  at  all,  at  room  temperature. 

Upon  Loeffler's  Hood  serum,  colonies  appear  after  twenty-four  to 
thirty-six  hours  as  small  indentations  which  indicate  a  liquefaction 
of  the  medium.  Axenfeld  states  that  eventually  the  entire  medium 

6  Wollstein,  loc.  cit. 

7  Morax,  Ann.  de  1'inst.  Pasteur,  1896. 

8  Axenfeld,  Cent.  f.  Bakt,,  xxi,  1897. 


ZUR  NEDDEN'S  BACILLUS  509 

may  become  liquefied.    Upon  serum  agar  delicate  grayish  drop-like 
colonies  are  formed  which  are  not  unlike  those  of  the  gonococcus. 

In  ascitic   bouillon  general  clouding  occurs  within  twenty-four 
hours. 


FIG.  54.— MORAX-AXENFELD  DIPLO-BACILLUS. 

Pathogenicity. — Attempts  to  produce  lesions  in  the  lower  animals 
with  this  bacillus  have  been  universally  unsuccessful.  Subacute  con- 
junctivitis, however,  has  been  produced  in  human  beings  by  inocula- 
tions with  pure  cultures. 


ZUR  NEDDEN'S  BACILLUS 

In  ulcerative  conditions  of  the  cornea,  Zur  Nedden  has  frequently 
found  a  bacillus  to  which  he  attributes  etiological  importance. 

The  bacillus  which  he  has  described  is  small,  usually  less  than  one 
micron  in  length,  often  slightly  curved,  and  generally  found  singly. 
It  may  be  found  in  the  diplo  form  but  does  not  form  chains.  It  is 
stained  by  the  usual  dyes,  often  staining  poorly  at  the  ends.  Stained 
by  Gram's  method  it  is  decolorized.  The  bacillus  is  non-motile. 

Cultivation. — It  is  easily  cultivated  upon  the  ordinary  laboratory 
media.  Tpon  agar  it  forms,  within  twenty-four  hours,  transparent, 
slightly  fluorescent  colonies  which  are  round,  raised,  rather  coarsely 
granular,  and  show,  a  tendency  to  confluence. 


510  PATHOGENIC  MICROORGANISMS 

Gelatin  is  not  liquefied. 
Milk  is  coagulated. 

Upon  potato,  there  is  a  thick  yellowish  growth. 
Upon  dextrose  media,  there  is  acid  formation,  but  no  gas. 
The  bacillus  forms  no  indol  in  pepton  solutions. 
Pathogenicity. — Corneal  ulcers  have  been  produced  by  inocula- 
tion of  guinea-pigs. 

/     BACILLUS  OF  DUCREY 

The  soft  chancre,-  or  chancroid,  is  an  acute  inflammatory,  de- 
structive lesion  which  occurs  usually  upon  the  genitals  or  the  skin 
surrounding  the  genitals.  The  infection  is  conveyed  from  one 
individual  to  another  by  direct  contact.  It  may,  however,  under 
conditions  of  surgical  manipulations,  be  transmitted  indirectly  by 
means  of  dressings,  towels,  or  instruments. 

The  lesion  begins  usually  as  a  small  pustule  which  rapidly 
ruptures,  leaving  an  irregular  ulcer  with  undermined  edges  and  a 
necrotic  floor  which  spreads  rapidly.  It  differs  clinically  from  the 
true  or  syphilitic  chancre  in  the  lack  of  induration  and  in  its  violent 
inflammatory  nature.  Usually  it  leads  to  lymphatic  swellings  in  the 
groin  which,  later,  give  rise  to  abscesses,  commonly  spoken  of  as 
"buboes." 

In  the  discharges  from  such  lesions,  Ducrey,9  in  1889,  was  able 
to  demonstrate  minute  bacilli  to  which  he  attributed  an  etiological 
relationship  to  the  disease,  both  because  of  the  regularity  of  their 
presence  in  the  lesions  and  the  successful  transference  of  the  disease 
by  means  of  pus  containing  the  microorganisms. 

Morphology  and  Staining. — The  Ducrey  bacillus  is  an  extremely 
small  bacillus,  measuring  from  one  to  two  micra  in  length  and  about 
half  a  micron  in  thickness.  It  has  a  tendency  to  appear  in  short 
chains  and  in  parallel  rows,  but  many  of  the  microorganisms  may 
be  seen  irregularly  grouped.  It  is  not  motile,  possesses  no  flagella, 
and  does  not  form  spores. 

Stained  by  the  ordinary  anilin  dyes,  it  has  a  tendency  to  take  the 
color  irregularly  and  to  appear  more  deeply  stained  at  the  poles.  By 
the  Gram  method,  it  is  decolorized.  In  tissue  sections,  it  may  be 
demonstrated  by  Loeffler's  methylene-blue  method,  and  in  such 
preparations  has  been  found  within  the  granulation  tissues  forming 

*  Ducrey,  Monatschr.  f.  prakt.  Dermal.,  9,  1889. 


BACILLUS  OF  DUCREY  511 

the  floor  of  the  ulcers.  In  pus,  the  bacilli  are  often  found  within 
leucocytes. 

Cultivation  and  Isolation. — Early  attempts  at  cultivation  of  this 
bacillus  were  universally  unsuccessful  in  spite  of  painstaking  experi- 
ments with  media  prepared  of  human  skin  and  blood  serum.  In  1900, 
Besanc,on,  Griffon,  and  Le  Sourd 10  finally  succeeded  in  obtaining 
growths  upon  a  medium  containing  agar  to  which  human  blood  had 
been  added.  They  were  equally  successful  when  dog's  or  rabbit's 
blood  was  substituted  for  that  of  man.  Since  the  work  by  these 
authors,  the  cultivation  by  similar  methods  has  been  carried  out  by 
a  number  of  investigators.  Coagulated  blood,  which  has  been  kept 
for  several  days  in  sterile  tubes,  has  been  found  to  constitute  a  favor- 
able medium.  Freshly  clotted  blood  cannot  be  employed,  probably 
because  of  the  bactericidal  action  of  the  serum.  Serum-agar  has 
occasionally  been  used  with  success,  but  does  not  give  results  as 
satisfactory  as  those  obtained  by  the  use  of  the  whole  blood. 

The  best  method  of  obtaining  pure  cultures  upon  such  media 
consists  in  puncturing  an  unruptured  bubo  with  a  sterile  hypodermic 
needle  and  transferring  the  pus  in  considerable  quantity  directly  to 
the  agar.  If  possible,  the  inoculation  of  the  media  should  be  made 
immediately  before  the  pus  has  had  a  chance  to  cool  off  or  to  be 
exposed  to  light.  When  buboes  are  not  available,  the  primary  lesion 
may  be  thoroughly  cleansed  with  sterile  water  or  salt  solution,  and 
material  scraped  from  the  bottom  of  the  ulcer  or  from  beneath  its 
overhanging  edges  with  a  stiff  platinum  loop.  This  material  is  then 
smeared  over  the  surface  of  a  number  of  blood-agar  plates. 

Upon  such  plates,  isolated  colonies  appear,  usually  after  forty- 
eight  hours.  They  are  small,  transparent,  and  gray,  and  have  a 
rather  firm,  finely  granular  consistency.  The  colonies  rarely  grow 
larger  than  pinhead  size,  and  have  no  tendency  to  coalesce.  At  room 
temperature,  the  cultures  die  out  rapidly.  Kept  in  the  incubator, 
however,  they  may  remain  alive  and  virulent  for  a  week  or  more. 

On  the  simpler  media,  glucose-agar,  broth,  or  gelatin,  cultivation 
is  never  successful.  On  moist  blood-agar  and  in  the  condensation 
water  of  such  tubes,  the  bacilli  have  a  tendency  to  grow  out  in  long 
chains.  Upon  media  which  are  very  dry,  they  appear  singly  or  in 
short  chains. 

During   recent   years   interest   has   again   been   aroused   in   the 

10  Besangon,  Griffon,  et  Le  Sourd,  Presse  med.,  1900. 


512  PATHOGENIC   MICROORGANISMS 

chancroidal  lesions  because  of  the  apparent  relative  frequency  of 
such  lesions  among  venereally  infected  soldiers  in  Europe.  We  are 
informed  by  Walker  that  during  the  post-armistice  periods  of  the 
existence  of  American  troops  in  France,  the  proportion  of  chancroids 
to  other  venereal  infections  rose  quite  beyond  the  ordinary  relative 
proportion  of  this  variety  of  infection,  apparently  for  the  reason 
that  prophylaxis  as  practiced  had  less  effect  upon  chancroidal  infec- 
tion than  it  did  upon  the  syphilitic  and  gonorrheal  infections.  Since 
there  had  apparently  developed  in  the  minds  of  genito-urinary 
specialists,  a  certain  amount  of  skepticism  regarding  the  role  played 
by  the  Ducrey  bacillus  at  this  time,  the  matter  was  reinvestigated 
in  our  laboratory  by  Teague  and  Deibert.11  They  developed  a 
method  for  direct  diagnostic  cultivation  of  Ducrey  bacilli  from  chan- 
croidal lesions  which  has  so  much  practical  value  that  it  will  be  well 
to  quote  it  in  considerable  detail.  The  method  as  described  by  them 
is  as  follows:  A  rabbit  is  bled  from  the  heart  with  a  sterile  20  c.c. 
syringe  and  the  blood  is  distributed  in  amounts  of  1  c.c.  in  small  test 
tubes,  a  little  larger  than  the  ordinary  Wassermann  tube.  The  blood 
is  allowed  to  clot  at  room  temperature  and  is  then  heated  for  five 
minutes  at  55°  C.  It  can  thus  be  preserved  in  the  ice-box  or  can  be 
used  immediately.  Equally  good  results  can  be  obtained  when  the 
tubes  are  kept  in  the  ice-box  for  three  to  four  days  before  use  with- 
out heating. 

Pieces  of  stiff  iron  wire,  gauge  18,  about  5y2  inches  long  are  bent 
upon  themselves  at  one  end  for  about  %  inch.  Ten  or  twelve  of 
these  wires  are  placed  in  a  6-inch  test  tube  and  are  heated  in  the 
dry  sterilizer.  The  patient  removes  the  dressing  and  a  bit  of  the  pus 
is  picked  up  with  the  bent  end  of  the  wire,  the  latter  having  been 
first  rubbed  gently  over  the  base  of  the  ulcer  or  under  its  undermined 
edge.  The  pus  is  then  transferred  to  a  tube  of  clotted  blood  and 
distributed  in  the  serum  by  passing  the  wire  around  the  clot.  A 
second  tube  is  prepared  in  the  same  way.  After  twenty-four  hours 
incubation  at  37°  C.  the  serum  around  the  clot  is  thoroughly  stirred 
with  a  platinum  loop  and  a  smear  is  made.  Examination  with  the 
oil-immersion  lens  shows  characteristic  chains  of  small  Gram- 
negative  bacilli,  sometimes  in  pure  culture,  sometimes  in  mixed  cul- 
ture. The  organism  is  usually  so  characteristic  that  such  an  exam- 
ination is  sufficient  basis  for  a  positive  diagnosis.  Even  when  anti- 

11  league  and  Deibert,  Jour,  of  Urology,  4,  1920,  543. 


BACILLUS  OF  DUCREY  513 

septic  power  or  ointments  have  been  applied,  repeated  positive  cul- 
tures have  been  obtained  by  finding  a  bit  of  pus  free  from  drug.  It 
is  not  even  necessary  to  wash  the  ulcer  before  taking  cultures. 

At  the  time  of  the  publication  of  their  first  paper,  Teague  and 
Deibert  had  cultured  by  the  above  method,  274  sores.  In  most  cases 
these  were  indiscriminately  cultured,  even  in  many  cases  when  no 
clinically  characteristic  picture  was  apparent.  Of  these  274  sores, 
140  yielded  positive  Ducrey  cultures.  Of  the  134  negative  cases, 
satisfactory  notes  were  obtained  of  only  69,  and  from  these  notes  it 
is  apparent  that  42  of  these  69  negative  cases  at  least  were  not 
chancroidal  but  primary  syphilitic  lesions.  It  seems  to  Teague  fair 
to  assume  that  by  this  method  probably  over  90  per  cent  of  true 
chancroids  can  be  diagnosed,  and  it  is  so  simple  that  the  physician 
in  the  clinic  can  take  the  cultures  as  directed  and  send  them  to  the 
laboratory.  Isolations  can  subsequently  be  made  by  inoculating 
blood  agar  plates  from  the  clotted  blood  tubes  after  24  hours.  The 
nutrient  agar  should  have  a  PH  of  7.2  or  7.3,  and  the  agar  must  be 
neither  too  stiff  nor  its  surface  too  dry.  Teague 's  results  not  only 
furnish  a  simple  method  for  the  determination  of  mixed  infection, 
but  also  reaffirm  the  etiological  importance  of  the  Ducrey  bacillus  in 
chancroids. 

As  to  prophylactic  treatment,  the  recent  experience  seems  to 
indicate  that  warm  water  and  soap  very  thoroughly  applied  is  prob- 
ably more  effective  in  the  prophylaxis  of  this  type  of  infection,  than 
are  the  specific  methods  used  for  venereal  prophylaxis. 

Pathogenicity. — Besanc,ori,  Griffon,  and  Le  Sourd,  and  others, 
have  succeeded  in  producing  lesions  in  man  by  inoculation  with  pure 
cultures.  Inoculation  of  the  lower  animals  has,  so  far,  been  entirely 
without  result. 


CHAPTER  XXVI 

MIOROCOCCUS  INTRACELLULARIS  MENINGITIDIS   (MENINGOCOCCUS) 
AND  EPIDEMIC  CEEEBBOSPINAL  MENINGITIS 

INFECTIOUS  processes  in  the  meninges  may  be  caused  by  many 
different  microorganisms. 

Meningitis  may  be  primary  or  secondary.  Secondary  meningitis 
may  often  occur  during  the  course  of  pneumonia,  when  pneumococci, 
carried  to  the  meninges  by  the  blood  stream,  give  rise  to  a  usually 
fatal  form  of  the  disease.  More  rarely  a  similar  process  may  occur 
as  a  secondary  manifestation  of  typhoid  fever  or  influenza.  Menin- 
gitis may  also  result  secondarily  by  direct  extension  from  sup- 
purative  lesions  about  the  skull,  such  as  those  occurring  in  diseases 
of  the  middle  ear  or  frontal  sinuses  or  after  compound  fractures.  In 
such  cases  the  invading  organisms  are  usually  staphylococci,  strepto- 
cocci, or  pneumococci. 

Isolated  cases  of  meningeal  infection  with  B.  coli,  B.  para- 
typhosus,  Bacillus  pestis,  and  Bacillus  mallei  have  been  reported- 
A  frequent  more  chronic  form  of  the  disease  is  caused  by  Bacillus 
tuberculosis. 

Primary  acute  meningeal  infection,  however,  is  due  chiefly  to 
two  microorganisms,  Micrococcus  intracellularis  meningitidis,  and 
the  pneumococcus. 

A  tabulation  of  the  comparative  frequency  with  which  the  vari- 
ous microorganisms  are  found  in  the  meninges  has  been  attempted 
by  Marschal.1  This  author  estimates  that  about  69.2  per  cent  of  all 
acute  cases  are  due  to  the  meningococcus,  20,8  per  cent  to  Diplo- 
coccus  pneumonias,  and  the  remaining  10  per  cent  to  the  other  bac- 
teria mentioned. 

The  cases  caused  by  the  pneumococcus  and  the  other  less  frequent 
incitants  usually  occur  sporadically.  When  the  disease  occurs  in 
epidemic  form,  it  is  almost  always  due  to  the  meningococcus. 

1  Marschal,  Diss.  Strassburg,  1901,  Quoted  from  Weichselbaum,  in  Kollc  und 
Wassermann,  ' '  Handbuch. ' ' 

514 


MICROCOCCUS    INTRACELLULARIS    MENINGITIDIS          515 

Diplococcus  intracellularis  meningitidis  was  first  seen  in  menin- 
geal  exudates  by  Marchiaf ava  and  Celli 2  in  1884.  These  authors 
not  only  described  accurately  the  morphological  characteristics  now 
recognized,  but  also  called  attention  to  the  intracellular  position  of 
the  microorganism  and  to  its  gonococcus-like  appearance.  They 
failed,  however,  to  cultivate  it. 

Observations  confirmatory  of  the  Italian  authors  were,  soon  after, 
made  by  Leichtenstern.3  Cultivation  and  positive  identification  as  a 
separate  species  was  not  accomplished,  however,  until  Weichsel- 
baum,4  in  1887,  reported  his  observations  upon  six  cases  of  epidemic 
cerebrospinal  meningitis  in  which  he  not  only  found  the  cocci  mor- 
phologically, but  was  able  to  study  their  biological  characteristics  in 
pure  culture.  The  researches  of  Weichselbaum  were  soon  confirmed 
and  extended  by  elaborate  studies  5  which  left  no  doubt  as  to  the 
specific  relationship  between  the  microorganism  cultivated  by  him 
and  the  clinical  condition. 

Morphology  and  Staining. — Stained  in  the  spinal  fluid  from  an 
infected  patient,  the  meningococcus  bears  a  striking  similarity  to 
the  gonococcus.  The  microorganisms  appear  intra-  and  extracellu- 
larly,  usually  in  diplococcus  groups,  sometimes  as  tetrads,  or  even  in 
larger  agglomerations.  The  individual  diplo-forms  are  flattened  on 
the  sides  facing  each  other,  presenting  somewhat  the  biscuit-form 
of  the  gonococcus.  The  variation  in  size  of  the  cocci  in  the  same 
smear  is  a  noticeable  feature  and  of  some  diagnostic  importance. 
This  dissimilarity  in  size  is  noticeable  also  in  cultures,  which, 
especially  when  older  than  twenty-four  hours,  contain  forms  double 
or  even  triple  the  size  of  the  average  coccus.  These  may  possibly  be 
involution  forms. 

The  meningococcus  is  non-motile  and  non-spore  forming.  It 
stains  easily  with  all  the  usual  aqueous  anilin  dyes.  Its  behavior 
toward  Gram's  stain  was  long  a  subject  of  controversy,  owing  to 
the  error  of  Jaeger,6  who  claimed  to  have  found  it  Gram-positive. 
There  is  no  question  now,  however,  that  the  cocci  decolorize  by 
Gram's  method  when  this  is  carefully  carried  out. 

2  Marchiaf  ava  and  Celli,  Gaz.  degli  ospedali,  8,  1884. 

3  Leichlenstern,  Dent.  med.  Woch.,  1885. 

4  Weichselbaum,  Fort.  d.  Med.,   1887. 

5  Councilman,  Mallory,  and  Wright,  Special  Eep.  Mass.  Board  of  Health,  1898, 
Albrecht  und  Ghon,  Wien.  klin.  Woch.,  1901. 

6  Jaeger,  Zeit.  f.  Hyg.,  xix,  1895. 


516 


PATHOGENIC   MICROORGANISMS 


In  spinal  fluid  satisfactory  preparations  may  be  obtained  by 
staining  in  Jenner  's  blood  stain.  Councilman,  Mallory,  and  Wright 7 
were  the  first  to  notice  that,  when  stained  with  Loeffler  's  methylene- 
blue,  meningococcus  stains  irregularly,  showing  metachromatic  gran- 
ules in  the  center  of  the  cell  bodies.  These  granules  can  be  demon- 
strated more  clearly  with  the  Neisser  stain  employed  for  similar 
demonstration  in  the  case  of  B.  dipththeriae  and  have  some  value  in 
differentiating  meningococcus  from  gonococcus. 


RT   'J 


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FIG.  55. — MENINGOCOCCUS  PURE  CULTURE. 

It  is  important  to  remember  that  meningococci  in  spinal  fluid 
undergo  solution  very  readily,  a  solution  which  is  probably  an 
autolysis,  with  the  result  that  spinal  fluid  which  may  be  full  of 
polymorphonuclear  leucocytes,  contains  very  few  recognizable  or- 
ganisms. This  readiness  of  meningococci  to  go  into  solution  will  be 
spoken  of  below  in  connection  with  problems  of  cultivation. 

Cultivation  of  the  Meningococcus. — The  meningococcus  is  peculiar 
in  that  there  is  considerable  difference  in  the  ease  with  which  separate 
strains  can  be  made  to  grow  upon  artificial  media.  Some  meningo- 
cocci grow  readily  upon  all  meat  infusion  culture  media.  They  may 
even  grow  upon  some  meat  extract  media,  but  growth  upon  these  is 
never  profuse.  It  is  never  well  to  rely  upon  media  to  which  no 
enriching  substance  has  been  added,  or  that  have  not  been  especially 
made  for  meningococcus  cultivation  when  attempts  are  made  at  first 


7  Councilman,  Mallory ,  and  Wright,  Eep.  Mass.  State  Bel.  of  Health,  1898. 


MICROCOCCUS    INTRACELLITLARIS    MENINGITIDIS          517 

isolation  from  human  material.  After  the  bacteriologist  is  familiar 
with  the  individual  strains,  he  may  at  times  carry  his  strains  on  the 
simpler  media,  meat  infusion  agar  and  broth. 

Growth  is  more  luxuriant  and  rapid  upon  media  to  which  animal 
protein  in  the  form  of  blood  serum  or  ascitic  fluid  has  been  added. 
Coagulated  serum  is  not  liquefied.  For  cultivation  of  the  meningo- 
coccus  directly  from  the  human  body  it  is  wise  to  use  the  richer  serum 
or  blood  media.  Agar  to  which  whole  rabbit's  blood  has  been  added 


FIG.  56. — MENINGOCOCCUS  IN  SPINAL  FLUID. 

forms  an  excellent  medium,  both  for  cultivation  and  for  keeping  the 
organism  alive.  Loeffler's  blood  serum  is  less  favorable.  It  is  advisable, 
too,  when  cultivating  directly  from  spinal  fluid,  to  plant  rather  large 
quantities  (1  to  2  c.c.),  since  many  of  the  cocci  in  the  exudate  will  fail 
to  develop  colonies,  possibly  because  of  their  prolonged  exposure  either 
to  the  body  fluids  or  to  their  own  products  in  a  closed  space. 

Upon  broth,  growth  is  slow  and  takes  place  chiefly  upon  the  sur- 
face, the  sediment  consisting  mainly  of  dead  bacteria.  Glucose  added 
to  agar  or  to  broth  renders  the  medium  more  favorable  for  rapid 


518  PATHOGENIC   MICROORGANISMS 

growth,  but,  owing  to  acid  formation,  tends  to  cause  a  more  rapid  death 
of  the  culture.  In  flasks  of  broth  containing  glucose  one  per  cent,  and 
CaC03  one  per  cent,  however,  cultures  have  been  kept  alive  for  as 
long  as  fourteen  months  (Hiss).  On  milk,  growth  takes  place  without 
coagulation  of  the  casein.  Potatoes  are  not  a  favorable  medium, 
though  growth  occasionally  takes  place. 

While  slight  alkalinity  or  acidity  does  not  inhibit,  the  most  favor- 
able reaction  of  media  is  a  Pw  of  7.4  to  7.6. 

H 

Oxygen  is  necessary  for  development.  Complete  anaerobiosis,  while 
not  absolutely  inhibitory,  is  extremely  unfavorable,  unless  proper 
carbohydrates  be  present. 

Recently,  work  by  Wherry  and  Erwin,8  as  well  as  by  Gates,9  has 
shown  that  the  growth  of  the  meningococcus  is  definitely  stimulated 
by  replacing  about  ten  per  cent  of  the  air  by  carbon  dioxide.  The 
plates  are  placed  into  a  closed  jar,  into  which  freshly  produced  carbon 
dioxide  is  allowed  to  pass,  and  the  jars  are  then  sealed  and  incubated. 
This  matter  has  been  discussed  in  a  previous  section  on  partial  oxygen 
tensiort  in  bacterial  cultures.  We  have  confirmed  this  in  our  labora- 
tory, and  find  that  the  colonies  grow  larger  and  growth  is  more  rapid 
under  the  partial  C02  atmosphere. 

While  growth  may  take  place  at  temperatures  ranging  from  25° 
to  42°  C.,  the  optimum  is  37.5°  C.  It  is  an  important  aid  to  the 
recognition  of  true  meningococci  that  they  never  grow  at  ordinary 
room  temperature.  Apart  from  the  remarkable  viability  displayed 
upon  calcium-carbonate  broth,  the  average  length  of  time  during 
whiclTthe  meningococcus  will  remain  alive  without  transplantation  is 
rather  short.  Recently  isolated  cultures  grown  on  agar  or  serum-agar 
may  die  within  two  or  three  days.  Accustomed  to  artificial  cultivation 
through  a  number  of  generations,  however,  the  cultures  become  more 
hardy  and  transplantation  may  safely  be  delayed  for  a  week  or  even 
longer.  Albrecht  and  Ghon10  have  kept  a  culture  alive  on  agar  for 
one  hundred  and  eighty-five  days.  It  is  a  strange  fact  that  after  pro- 
longed artificial  cultivation  some  strains  of  meningococcus  may  grad- 
ually lose  their  growth  energy  and  finally  be  lost  because  of  their 
refusal  to  develop  in  fresh  transplants. 

It  is  our  belief  that  this  phenomenon  which  hitherto  we  have  been 
at  a  loss  to  explain,  may  have  some  connection  with  the  so-called 


8  Wherry  and  Erwin,  Jour,  of  Infec.  Dis.,  22,  1918,  194. 

8  Gates,  Jour.  Exper.  Med.,  29,  1919,  325. 

10  Albrecht  und  Ghon,  Wien.  klin.  Woch.,  1901. 


MICROCOCCUS    INTRACELLULARIS    MENINGITIDIS  519 

"bacteriophage"  phenomena  discussed  in  a  separate  section.  Since 
the  extensive  investigation  of  the  autolytic  properties  acquired  by 
bacteria  under  certain  conditions,  there  has  been  no  investigation  of 
the  meningococcus  problem  from  this  point  of  view.  We  ourselves 
have  often  found  meningococcus  cultures  to  lose  their  viability  under 
conditions  which,  retrospectively,  we  suspect  now  may  have  been  due 
to  this  kind  of  development.  It  is  also  not  unlikely  that  the  extensive 
autolysis  of  the  organism  in  the  spinal  fluid  may  have  a  similar  sig- 
nificance. Storage  is  best  carried  out  at  incubator  temperatures.  At 
room  temperatures  or  in  the  ice  chest,  the  diplococcus  dies  rapidly.11 

Special  Meningococcus  Media. — For  the  routine  cultivation  of 
meningococcus,  there  are  certain  media  which  are  better  than  others 
and  which  are,  therefore,  described  in  this  section. 

As  a  basis  for  meningococcus  media,  we  like  to  use  hormone  agar 
or  hormone  broth,  or  trypagar  or  trypsinized  broth  as  described  in  the 
section  on  media.  To  these  basic  media  enriching  substances  are  added. 
The  necessity  for  these  enriching  substances  may  have  a  more  complex 
cause  than  the  simple  addition  of  nutrition,  since,  as  Lloyd  has  sug- 
gested, the  occasional  first  growth  on  simple  media  of  meningococci 
directly  from  the  human  body,  may  depend  upon  the  presence  of  a 
certain  amount  of  "vitamine"  furnished  by  the  animal  fluids  present 
in  the  exudate.  The  most  convenient  substances  to  add  to  these  media 
are  blood  in  one  form  or  another.  Many  different  varieties  of  blood 
are  favorable,  and  human,  horse,  or  rabbit  blood  can  be  most  con- 
veniently used.  The  blood  may  be  defibrinated  and  added  directly  in 
quantities  of  about  five  per  cent,  and  if  agar  for  plating  is  used, 
melted  agar  is  mixed  with  the  blood  and  thoroughly  shaken  just  as  the 
plates  are  poured.  Laked  blood  is  very  convenient,  and  may  be  pre- 
pared by  mixing  whole  blood  with  about  four  parts  of  sterile  distilled 
water.  This  laked  blood  may  be  kept  and  mixed  with  the  agar  just 
before  pouring  the  plates,  after  the  agar  has  been  cooled  below  50°. 
The  blood  may  also  be  laked  in  ether  and  in  this  way  can  be  kept  sterile 
for  a  long  time  before  being  added  to  the  basic  medium.  Blood  serum 
and  ascitic  fluid  can  be  used,  but  do  not  seem  to  give  as  good  results  as 
does  laked  or  whole  blood.  The  addition  of  one-half  to  one  per  cent 
of  glucose  is  always  favorable.  The  special  pea-powder-blood-agar 

11 A  very  thorough  biological  study  of  meningococcus  and  related  organisms 
lias  recently  been  made  by  Elser  and  IFuntoon  (Jour.  Med.  Res.,  N.  S.  vol.  xv, 
1909),  which  may  be  consulted  for  a  more  detailed  description  of  cultural 
characteristics. 


520  PATHOGENIC  MICROORGANISMS 

used  by  the  British  during  the  war  is  not  described  in  detail  because 
we  believe  that  for  ordinary  laboratory  work  its  production  is  too  com- 
plicated, without  offering  sufficient  advantages  over  other  media.  To 
summarize,  we,  therefore,  recommend  for  plating  media,  carrier  work, 
and  isolation  from  spinal  fluid,  hormone  or  trypagar  with  a  PH  of 
7.4  to  7.5,  containing  one-half  per  cent  glucose,  to  which  about  5  or 
10  per  cent  of  defibrinated  or  laked  blood  is  added  just  before  the 
plates  are  poured. 

For  the  storage  of  stock  cultures,  Vedder's  starch  agar  described 
in  the  section  on  media  has  been  used  with  satisfaction. 

Gordon  12  and  others  also  have  used  coagulated  egg  media  in  slants 
for  storage  of  stock  cultures  with  good  results. 

Egg -yolk  Medium  for  the  Storage  of  Meningococcus  Cultures.13 — 
The  egg-yolk  used  may  be  the  yolks  of  eggs,  the  whites  of  which  have 
been  used  for  clearing  media.  One  volume  of  the  egg-yolk  is  mixed 
with  one-half  volume  of  physiological  salt  solution.  The  yolk  and 
salt  are  thoroughly  mixed,  tubed  and  slanted.  The  slants  are  then 
inspissated  in  the  usual  way.  This  can  be  done  in  an  autoclave  by 
bringing  the  temperature  up  gradually  without  letting  out  the  air, 
until  14  pounds  pressure  has  been  reached,  and  then  maintaining  this 
for  twenty  minutes.  Great  care  should  be  taken  to  prevent  bubbles 
in  the  medium.  The  tubes  should  be  plugged  with  paraffin,  since  water 
of  condensation  is  necessary  to  make  the  medium  useful  for  storage. 

For  fermentation  reactions,  solid  or  fluid  media  with  various 
sugars  and  litmus  indicator,  may  be  used.  Gordon,  whose  experience 
in  this  kind  of  work  has  been  extensive,  used  for  his  fermentations  a 
liquid  medium  of  simple  pepton  water  with  one  per  cent  blood  serum 
and  the  sugar  to  be  investigated,  added.  Elser  and  Huntoon  14  em- 
ployed among  other  things,  for  their  extensive  fermentation  work, 
sheep  serum  water,  ascitic  broth,  and  broth  made  with  nutrose. 

Resistance. — The  meningococcus  is  killed  by  exposure  to  sunlight 
or  to  drying  within  twenty-four  hours.15  It  is  extremely  sensitive 
to  heat  and  cold  and  by  the  common  disinfectants  is  killed  in  high 
dilutions  and  by  short  exposures,  At  0°  C.  it  usually  dies  within  two 
or  three  days. 


^-Gordon,   FlacTc   and    Hinex,   Med.   Kes.    Com.,   Special    Report    Series,   No.   3, 
London,    1917. 

"Directions  of  Major  Foster  from  Gordon's  Laboratory. 

14  Elser  and  Huntoon,  Jour.  Med.  Eos.,  20,  1909,  371. 

15  Councilman,  Mallory,  and  Wright,  Boston,  1898;  Albrecht  and  Ghon,  loc.  cit. 


MICROCOCCUS    INTRACELLULARIS    MENINGITIDIS          521 

A  special  study  of  the  resistance  of  meningococci  to  various 
dye  stuffs  has  been  made  by  Binger.16  An  interesting  result  of 
these  investigations  was  that  Binger  found  that  methylene-blue 
had  a  specific  inhibiting  action  upon  meningococcus  and  gonococcus 
at  dilutions  too  IOAV  to  inhibit  other  pathogenic  microorganisms,  and 
that  this  inhibitory  action  was  not  interfered  with  by  the  presence 
of  the  protein  in  spinal  fluid  or  other  exudates. 

Toxic  Products  of  the  Meningococcus. — No  soluble  exotoxin  has  ever 
been  conclusively  isolated  from  meningococcus  cultures.  A  substance 
which  causes  acute  symptoms  in  rabbits  within  an  hour  can  be  recovered 
from  young  broth  cultures  of  meningococci,  and  from  filtered  wash- 
ings from  meningococcus  cultures  on  agar.  These  substances  are 
analogous  to  the  so-called  "X"  substances  which  one  of  the  writers 
with  Kuttner  and  Parker17  has  described,  which  are  non-specific,  can 
be  obtained  from  many  different  microorganisms  and  are  non- 
antigenic.  That  they  are  a  very  real  and  important  substance  in 
connection  with  meningococci  we  are  persuaded  to  believe  by  the  fact 
that  those  who  immunize  horses  for  serum  production  with  meningo- 
cocci find  that  it  is  necessary  to  wash  the  agar  cultures  once  before 
injecting  into  horses,  otherwise  severe  symptoms  occasionally  result. 

A  number  of  investigators  have  found  that  cultures  that  have  been 
kept  in  broth  long  enough  for  a  certain  amount  of  extraction  or 
autolysis  to  occur,  yield  toxic  products  which  are  in  general  identical 
in  their  action  to  that  of  whole  meningococci  injected  in  analogous 
quantities.  This  has  been  the  experience  of  Flexner,18  Kraus  and 
Doerr,19  and  others.  Extracts  of  meningococci  made  with  salt  solution, 
weak  sodium  hydrate,  etc.,  kill  guinea  pigs  within  24  hours,  with 
symptoms  of  general  intoxication,  peritoneal  exudates,  and  often 
pleural  exudates.  Intravenous  injection  of  sufficient  quantities  of  such 
extracts  or  of  dead  meningococci  may  kill  rabbits.  No  reliable  or  con- 
stant results  with  such  substances  have  been  obtained,  but  it  is  quite 
definite  that  the  bodies  of  meningococci,  like  the  bodies  of  typhoid  and 
colon  bacilli,  are  toxic  for  animals  by  what  is  generally  spoken  of  as  an 
endotoxin  action. 


16  Binger,  Jour.  Infec.  Dis.,  25,  1919,  277. 

17  Zinsser,  Kuttner  and  Parker,  Proc.  Soc.  Exper.  Biol.  and  Med.,  November, 
1920. 

18  Flexner,  Cent,  f .  Bakt.,  43,  1907. 

19  Kraus  and  Doerr,  Wien.  klin.  Woch.,  1908. 


522  PATHOGENIC   MICROORGANISMS 

Types  of  Meningococci. — Until  1909  it  was  believed  that  the  men- 
ingococcus  group,  was  homogeneous,  and  that  no  essential  difference 
between  individual  members  of  the  group  existed.  In  this  year, 
Dopter  20  found  that  some  of  the  meningococci  isolated  from  cases 
which  occurred  in  Paris  and  environment,  could  be  distinguished  "by 
specific  agglutination  reactions  from  the  ordinary  or  normal  type. 
This  para-meniiigococcus,  as  Dopter  called  it,  opened  the  way  for 
investigations  aimed  at  the  serological  classification  of  the  group,  and, 
as  was  to  be  expected,  it  was  found  that  there  were  a  considerable 
number  of  different  meiiingococcus  sub-types.  Wollstein  21  confirmed 
Dopter 's  work  and  found,  among  other  things,  that  the  various  para- 
meningococeus  strains  were  not  wholly  homologous,  and  suggested 
their  possible  further  subdivision.  Gordon  22  examined  a  large  num- 
ber of  meningococci  from  cases  occurring  among  British  and  Canadian 
troops,  and  found  that  all  the  organisms  studied  by  him  could  be 
divided  into  four  definite  types.  He  used  not  only  the  agglutination 
reaction,  but  controlled  them  with  absorption  tests.  Tulloch,23  follow- 
ing up  Gordon's  work  on  a  considerable  material,  found  that,  out  of 
356  cocci  investigated,  234  gave  specific  results  with  the  four  type  sera 
used  by  Gordon's  laboratory.  He  found  that,  with  remarkably  few 
exceptions,  the  organisms  responsible  for  the  outbreaks  among  British 
troops  were  comprised  in  the  four  Gordon  types.  He  did,  however, 
find  some  organisms  in  the  nasopharyngeal  cultures  of  carriers  which, 
though  closely  resembling  meningococci,  did  not  react  with  any  of  the 
type  sera.  There  was  some  question,  however,  in  his  mind  as  to 
whether  these  represented  true  virulent  meningococci.  An  important 
result  of  Gordon's  investigations  was  to  show  that  very  many  of  the 
organisms  obtained  from  carriers  belong  to  one  of  the  four  types 
known  to  exist  in  actual  cases  of  meningeal  infection. 

In  America,  Flexner 24  and  his  associates  have  investigated  the 
group  relationships  of  the  meningococci  very  carefully,  and  their 
results  indicate  that  there  are  probably  two  main  types,  the  normal 
and  the  para-meningococcus  of  Dopter;  and,  in  addition  to  this,  a 
considerable  number  of  heterogeneous  intermediate  types  which  are 
related  to  each  other  and  to  the  fixed  types  more  or  less  in  the  same 

20  Dopter,  Cdmpt.  Eend.  de  la  Soc.  de  Biol.,  67,  1909,  74. 

21  Wollstein,  Jour.  Exper.  Med.,  20,  1914. 

22  Gordon,  Brit.  Med.  Res.  Commit.  Eeports,  London,  1915  and  1917. 
28  Tulloch,  Jour.  Royal  Medical  College,  February,  1918,  p.  9. 

24  Flexner,  Bulletin,  Rock.  Inst.  for  Med.  Res.,  1917. 


MICROCOCCUS    INTRACELLULARIS    MEN1NGITIDIS  523 

way,  but  somewhat  more  closely  than  are  the  different  members  of  the 
viridans  group  of  streptococci,  a  point  which  makes  it  plain  that  a 
diagnostic  or  curative  serum,  to  be  truly  polyvalent,  must  be  produced 
with  many  different  representatives  of  organisms  isolated  from  cases. 
The  correspondence  of  the  different  types,  as  named  in  various  coun- 
tries, is  as  follows: 

Gordon's  type      I  =  para-meningococcus 

Gordon's  type     II  =  normal  meningococcus 

Gordon's  types  III  and  IV     =  intermediate  or  irregular  strains, 

of  which  there  are  a  considerable  number  of  different  ones, 

shading  into  each  other,  serologically. 

As  far  as  the  prevalence  of  type  is  concerned,  no  definite  rule  can 
be  established  at  present.  In  the  extensive  investigations  of  Gordon 
and  his  co-workers,  it  was  found  that  the  earliest  cases  were  mostly  his 
type  I,  later  came  his  type  II,  especially  in  the  London  district,  and 
after  March  type  IV  cases  began  to  appear,  but  no  type  III  cases  were 
noticed  until  July. 

Agglutination. — Immunization  of  animals  by  repeated  inocula- 
tions of  meningococcus  25  results  in  the  formation  in  the  blood  serum 
of  agglutinins.  Kolle  and  Wassermann  26  obtained  from  horses  a 
serum  which  had  an  agglutinating  value  of  1 :3,000  for  the  homol- 
ogous strain,  and  of  as  much  as  1 :500  for  other  true  meningococcus 
strains.  Similar  experiments  by  Dunham27  and  others  have  proved 
the  unquestionable  value  of  agglutination  for  species  identification 
of  this  group.  Great  differences  may,  however,  exist  between  indi- 
vidual races  in  their  agglutinability  in  the  same  immune  serum. 

Kutscher  has  recently  called  attention  to  the  fact  that  strains 
which  cannot  be  'agglutinated  in  specific  sera  at  37°  C.  will  often 
yield  positive  results  when  subjected  to  55°  C.,  a  fact  of  some  prac- 
tical importance  if  confirmed. 

Elser  and  Huntoon  28  have  shown  that  in  the  serum  of  infected 
human  subjects  agglutination  of  some  strains  takes  place  in  dilutions 
as  high  as  1 :400. 

The  Production  of  Agglutinating  Sera  for  Meningococcus  Deter- 
mination in  •  Laboratories. — For  this  purpose,  rabbits  are  best 


K  Albreclit  and  Ghon,  Wien.  klin.  Woch.,   1901. 

26  Kolle  und  Wassermann,  Deut.  med.  Woch.,   15,   1906. 

27  Dunham,  Jour.  Inf.  Dis.,  11,  1907. 

28  Elser  and  Huntoon,  loc.  cit. 


524  PATHOGENIC   MICROORGANISMS 

.employed.  Amoss  has  found  that  young  rabbits  are  more  satisfac- 
tory than  older  ones  for  this  purpose,  and  he  uses  rabbits  weighing 
between  1500  and  1800  grams.  He  grows  his  meningococci  on  glu- 
cose agar  slants,  and  washes  up  the  growth  in  salt  solution.  0.001 
of  a  culture  is  inoculated  as  the  first  dose.  For  the  rapid  production 
of  agglutinating  sera,  he  injects  his  rabbits  for  three  succeeding 
days,  giving  a  rest  of  five  days,  and  then  another  course  of  three 
days  injection.  He  bleeds  the  animal  two  or  three  days  after  the 
second  course  of  inoculation.  For  ordinary  purposes,  the  slow 
method  of  three  or  four  day  intervals,  about  five  or  six  injections, 
with  bleeding  eight  or  nine  days  after  the  last  injection,  may  be 
used.  Among  English  workers,  Hine  29  injects  culture  suspensions 
grown  on  25  per  cent  hemoglobin  serum  agar,  killed  at  65°  and 
brought  to  a  standard  opacity.  0.5  per  cent  carbolic  acid  is  added 
for  preservation.  He  standardizes  all  his  suspensions  by  opacity 
comparisons  against  suspensions  of  freshly  precipitated  barium 
sulphate.  He  compares  by  diluting  his  suspension  in  a  test  tube  of 
similar  dimensions  as  the  standard  tube,  until  the  image  of  a  small 
flame  is  just  visible  in  the  same  distance  from  the  flame  as  in  the 
case  of  the  standard  tube.  With  such  suspensions  he  immunizes  rab- 
bits, beginning  with  an  injection  of  two  doses  of  five  hundred 
million  cocci  at  an  interval  of  one  hour.  Six  days  later  he  gives  three 
million  cocci,  and,  if  the  serum  is  satisfactory  on  the  eighth  day 
later,  he  bleeds.  This  method  was  satisfactory  in  the  hands  of  Hine, 
with  types  I  and  III.  With  the  other  types  he  has  had  to  give  larger 
and  more  frequently  repeated  doses.  In  all  such  immunization, 
experience  and  judgment,  with  frequent  titration  of  samples  of  the 
rabbit  serum  taken  from  the  ear,  are  necessary. 

Nicolle  30  at  the  Pasteur  Institute  uses  for  immunization,  pow- 
dered meningococcus  antigen  prepared  from  growth  on  agar  slants 
by  suspension  in  salt  solution,  centrifugation  and  drying.  Hine  also 
recommends  the  use  of  rabbits  ranging  from  800  to  1500  grams. 

Agglutination  Technique  ivith  Meningococci. — Agglutination  of 
meningococci  present  considerable  difficulties  because  of  the  relative 
inagglut inability  of  many  meningococcus  cultures.  This  is  a  peculiar- 
ity of  these  organisms  which  has  necessitated  much  investigation  and 
many  technical  modifications.  Hine  has  found  that  allowing  the 


29  Hinc,  Med.  lies.  Committee,  Spec.  Eep.,  Series  3,  No.  3,  p.  99, 

30  Nicolle, 


MICROCOCCUS    INTRACELLULARLS    MENINGITIDIS          525 

diluted  carbolic  saline  suspension  to  stand  for  twenty-four  hours, 
increases  agglutinability,  and  recommends  this  technique  if  time  per- 
mits. Tulloch31  has  called  attention  to  a  number  of  precautions  both 
in  handling  of  the  cultures  and  the  serum  for  agglutination  which  seem 
to  us  sufficiently  important  to  note.  He  recommends  the  use  of 
standardized  suspensions  of  the  meningococci  as  described  by  Hine, 
and  recommends  great  caution  in  the  nature  of  the  medium  on  which 
the  cultures  are  grown.  He  advises  getting  rid  of  the  condensation 
water  in  the  slants  before  washing  off  the  growth,  owing  to  the  possi- 
bility of  alkaliii  or  acid  reactions  in  this  condensation  water.  The 
strength  of  the  phenol  in  standard  suspensions  should  never  be  more 
than  0.5  per  cent.  Also  he  warns  against  getting  any  of  the  agar  into 
the  suspension  because  he  believes  that  it  may  act  under  certain  cir- 
cumstances as  a  protective  colloid. 

On  the  basis  that  moderate  heat  increases  the  agglutinability  of 
organisms  like  the  typhoid  bacillus  and  meningococci,  the  workers  in 
the  British  Central  Laboratory  used  the  routine  method  described 
under  carrier  determination,  that  is,  incubating  the  serum  culture 
agglutination  mixtures  in  a  water  bath  at  55°,  for  varying  periods, 
usually  twelve  hours  before  the  final  readings  are  made.  Hot  air  ovens 
at  55°  are  not  good  substitutes  because  of  the  great  evaporation  which, 
according  to  Hine,  occasionally  leads  to  spontaneous  agglutination. 
(See  also  section  on  Carriers.) 

A'gglutinin  Absorption  Experiments  for  Meningococcus  Typing.32 — 
A  thick  suspension  of  the  meningococci  to  be  examined,  quantity 
0.5  c.c.,  is  mixed  with  the  various  monovalent  type  sera,  0.5  e.c.,  dilu- 
tions 1  to  25,  in  saline.  Similar  serum  dilutions  were  set  up  without 
suspensions.  The  tubes  are  thoroughly  shaken,  set  in  the  water  bath  at 
37°  for  one  hour,  and  then  at  room  temperature  overnight.  The  tubes 
are  then  centrifugalized  at  high  speed  until  the  supernatant  fluid  is 
clear.  The  supernatant  fluids  of  the  tubes  containing  the  suspensions, 
as  well  as  the  fluids  similarly  treated  without  suspension,  now  repre- 
sent serum  dilutions  of  1  to  50. 

Each  suspension-absorption  tube  now  has  a  serum  control  which 
has  been  exposed  to  the  same  temperature  in  the  same  dilutions  with- 
out meningococci.  From  each  set  of  two  tubes  now  other  tubes  are 
made,  into  which  0.4,  0.2,  0.1,  and  0.05  c.c.  are  taken,  and  with  salt 

31  Tulloch,  Royal  Army  Med.  Jour.,  February,  1918. 

32  The  description  given   is  that  given  by  the  British  Medical  Research  Com- 
mittee, loc.  cit. 


526  PATHOGENIC   MICROORGANISMS 

solution  the  volume  of  all  of  these  tubes  is  brought  up  0.4  c.c.  To 
each  of  the  tubes  now  0.4  c.c.  of  the  homologous  meningococcus  sus- 
pension is  added,  giving  serum  dilutions  ranging  from  1  to  100,  to  1 
to  800.  These  are  now  incubated  at  55°  for  twenty-four  hours,  as  in 
the  first  agglutination.  The  agglutinating  titers  of  the  absorbed  sera 
are  now  compared  with  those  of  the  un-absorbed,  and  diminutions  of 
titer  are  noted. 

Animal  Pathogenicity. — Animals  are  not  very  susceptible  to 
infection  with  Diplococcus  meningitidis.  Subcutaneous  inoculation 
is  rarely  followed  by  more  than  a  local  reaction  unless  large  quan- 
tities are  used.  White  mice  are  rather  more  susceptible  than  other 
species.  Intraperitoneal  and  intravenous  inoculation  of  sufficient 
quantities  usually  results  in  the  death  of  mice,  rabbits,  guinea-pigs, 
and  dogs.  Occasional  strains  have  been  found  to  possess  a  not 
inconsiderable  degree  of  toxicity  for  rabbits,  grave  symptoms  or 
even  death  following  intravenous  injection  of  but  moderate  quanti- 
ties without  any  traceable  development  of  the  microorganisms  in  the 
organs  of  the  animals. 

Similar  observations  have  been  made  by  Albrecht  and  Ghon,33 
who  succeeded  in  killing  white  mice  with  dead  cultures.  It  would 
seem,  therefore,  that  the  effect  of  this  coccus  upon  animals  depends 
chiefly  upon  the  poisonous  substances  contained  in  the  bacterial 
bodies  (endotoxins).  Lepierre  34  has  obtained  the  meningococcus 
toxin  by  alcohol  precipitation  of  broth  cultures. 

Weichselbaum  himself  succeeded  in  producing  meningeal  sup- 
puration and,  in  one  case,  brain  abscess,  by  subdural  inoculation  of 
dogs.  Councilman,  Mallory,  and  Wright  produced  a  disease  in  many 
respects  similar  to  the  human  disease  by  intraspinous  inoculation  of 
a  goat.  More  recently,  Flexner  35  has  succeeded  in  producing  in 
monkeys  a  condition  entirely  analogous  to  that  occurring  in  human 
beings. 

THE  DISEASE  IN  MAN 

The  disease  produced  in  man  consists  anatomically  in  a  suppurative 
lesion  of  the  meninges,  involving  the  base  and  cortex  of  the  brain  and 
the  surface  of  the  spinal  cord.  The  nature  of  the  exudate  may  vary 

33  Albrecht  nnd  Ghon,  loc.  cit. 

"Lepierre,  Jour,  de  phys.  et  de  path,  gen.,  v,  No.  3. 

85  Flexner,  Journ.  of  Exp.  Med.,  1906. 


MICROCOCCUS    INTRACELLULARIS    MENINGITIDIS  527 

from  a  slightly  turbid  serous  fluid  to  that  of  a  thick  fibrinous  exudate. 
In  chronic  cases  encephalitis  and  dilatation  of  the  ventricles  may  take 
place.  Apart  from  their  presence  in  the  meninges  and  in  the  naso- 
pharynx, meningococci  have  not  been  satisfactorily  demonstrated  in 
any  of  the  complicating  lesions  of  the  disease.  Reports  of  their  pres- 
ence in  the  conjunctivas,  in  the  bronchial  secretions  from  broncho-  or 
lobar  pneumonia,  and  in  otitis  media,  have  been  reported  but  are 
not  very  common. 

The  occurrence  of  this  microorganism  in  the  circulating  blood  of 
meningitis  cases  has  been  definitely  proved  by  Elser,36  who  found  it 
in  ten  cases. 

In  the  discussions  on  epidemiology,  below,  we  will  see  that  Her- 
rick  and  others  claim  that  the  meningococcus  is  probably,  in  the 
majority  of  cases,  in  the  blood  before  it  reaches  the  meninges,  making 
its  way  to  the  central  nervous  system  by  way  of  the  blood  stream 
rather  than  directly  along  the  lymphatics  at  the  base  of  the  skull. 
It  seems  fair  to  assume  from  blood  culture  evidence  that  this  cer- 
tainly happens  in  many  cases  even  though  it  may  not  be  the  rule. 
During  epidemics,  also,  there  are  occasional  cases  in  which  a  general 
septicemia  due  to  meningococci  occurs,  without  ever  giving  rise  to 
symptoms  pointing  to  meningeal  involvement.  These  cases  are 
always  violent  in  course,  usually  fatal  and  accompanied  by  a  profuse 
petechial  rash. 


BACTERIOLOGICAL    MANAGEMENT    OF    THE    MENINGITIS 
CASE  AND  SERUM  TREATMENT 

In  the  light  of  our  present  knowledge  of  the  bacteriology  and 
serum  treatment  of  epidemic  meningitis,  a  considerable  responsibil- 
ity rests  with  the  bacteriologist.  The  difference  between  recovery 
and  death  may  depend  directly  upon  the  speed  with  which  a  bac- 
teriological diagnosis  is  made  and  a  proper  management  of  the  serum 
treatment.  When  a  case  of  suspicious  fever  in  which  slight  stiffness 
of  the  neck,  and  a  developing  Kernig  sign  is  associated  with  the 
other  indications  of  an  acute  infection,  the  first  step  must  consist  of 
lumbar  puncture. 

A  sterile  lumbar  puncture  needle  is  thrust  into  the  spinal  canal, 
a  little  to  one  side  of  the  third  or  fourth  lumbar  space,  and  the  fluid 

36  E Iscr,  Jour.  Med.  Ees.,  xiv,  1906. 


528  PATHOGENIC   MICROORGANISMS 

which  is  always  under  some  pressure,  is  taken  directly  into  a  centri- 
fuge tube.  This  fluid  must  then  be  examined  as  above  indicated  in 
the  technical  section  on  spinal  fluid,  and  the  diagnosis  made.  If  pos- 
sible, a  smear  should  be  made  at  the  bed  side,  and  an  immediate  Gram 
stain  done  with  the  first  drop  of  fluid  that  flows.  In  this  way,  it 
may  be  possible  to  inject  the  first  dose  of  serum  immediately  after 
the  withdrawal  of  the  diagnostic  fluid. 

Examination  of  Spinal  Fluid. — The  spinal  fluid  of  meningococcus 
cases  is  slightly  turbid  in  the  very  early  periods,  becoming  increas- 
ingly purulent,  with  large  numbers  of  polymorphonuclear  leucocytes. 
In  some  cases  the  fluid  which  has  been  very  purulent  may  clear 
up  considerably,  and  then  become  purulent  again,  a  matter  probably 
dependent  upon  sacculation  in  parts  of  the  subarachnoid  space.  The 
fluctuations  in  the  nature  of  the  spinal  fluid  under  intraspinous 
serum  treatment  will  be  spoken  of  in  another  place.  A  certain 
amount  of  prognostic  information  can  be  obtained  from  the  spinal 
fluid  in  that  in  severe  cases  that  are  not  doing  well,  there  will  be 
a  considerable  number  of  organisms,  extracellular.  Ordinarily,  the 
majority  of  the  meningococci  are  intracellular.  Such  spinal  fluid 
should  be  taken  into  sterile  centrifuge  tubes,  brought  to  the  labora- 
tory without  delay,  slides  smeared  from  the  sediment,  and  stained 
by  Jenner  and  by  Gram.  It  is  important  to  remember  that  because 
of  the  extensive  autolysis  of  meningococci  in  the  fluid,  it  may 
under  circumstances  be  very  difficult  to  find  meningococci.  In  such 
cases,  if  prolonged  search  has  failed  to  reveal  organisms,  our  ex- 
perience has  taught  us  to  assume  that  purulent  fluid  from  a  case  of 
an  acute  meningitis  in  which  there  are  a  preponderance  of  poly- 
morphonuclear leucocytes  without  organisms  is  probably  " meningo- 
coccus "  in  origin.  Streptococcus  and  pneumococcus  fluids  invariably 
show  Gram-positive  cocci. 

The  fluid  should  be  cultured  upon  blood  agar  plates,  the  medium 
prepared  as  described  above.  It  is  well  to  inoculate  the  plates 
heavily  since  the  viable  organisms  present,  even  in  acute  fluids, 
may  be  relatively  few  in  numbers.  To  be  on  the  safe  side  it  is 
sometimes  well  too,  to  place  a  portion  of  the  fluid  in  the  original 
centrifuge  tube  in  the  incubator  for  three  or  four  hours  before 
inoculating  media  from  it. 

The  typing  of  meningococci  derived  from  spinal  fluid  is  desirable 
since  the  preliminary  injection  of  polyvalent  serum  upon  first  diag- 
nosis may  be  advantageously  followed  by  the  injection  of  type  sera, 


MICROCOCCUS    INTRACELLULARIS    MENINGITIDIS          529 

homologous  to  the  organisms  found  in  the  patient.  This  method  is 
impracticable  on  a  large  scale  since  so  many  types,  shading  into 
each  other,  are  possible  in  this  disease.  However,  the  method  is 
used  to  a  considerable  extent  in  France. 

As  stated  in  the  section  on  the  manner  of  entrance  of  the  menin- 
gococci  into  the  subarachnoid  space,  it  is  a  question  now  under 
discussion  whether  the  organisms  travel  along  the  lymphatics  to  the 
base  of  the  skull  directly,  or  whether  bacteriemia  precedes  menin- 
geal  infection.  It  is  well,  always,  in  cases  of  early  meningitis,  to 
take  blood  cultures.  In  taking  blood  cultures  it  is  best  to  inoculate 
hormone  glucose  broth  flasks  containing  not  less  than  100  c.c.  of 
culture  fluid  and  to  make  a  number  of  glucose  hormone  agar  plates 
with  varying  amounts  of  blood.  The  presence  of  menmgococci  in 
the  blood  is,  of  course,  an  indication  for  intravenous  as  well  as 
intraspinous  injection  of  serum,  a  procedure  which  is  in  our  opinion 
advisable  in  all  cases,  since  it  is  quite  likely  that  meningococcus 
septicemia,  constant  or  intermittent  is  a  regular  feature  of  the 
pathology  of  the  disease. 

Serum  Therapy  of  Meningitis. — During  recent  years,  attempts 
have  been  made  to  treat  epidemic  meningitis  by  injections,  subcu- 
taneous and  intraspinous,  of  meningococcus-immune  serum.  Wasser- 
mann,37  in  1907,  reported  results  of  such  treatment  in  one  hundred 
and  two  patients,  with  a  recovery  of  32.7  per  cent.  The  serum, 
manufactured  by  Wassermann  and  his  associates,  was  obtained  from 
horses  immunized  with  cultures  of  meningococcus  and  with  toxic 
meningococcus  extracts.  More  recently  Flexner  and  Jobling38  have 
used  a  similar  serum  in  the  United  States  with  apparently  excellent 
results.  The  serum,  in  Flexner 's  cases,  as  in  the  technique  first 
used  by  Jochmann,  is  injected  intraspinously  after  a  quantity  of 
spinal  fluid  had  been  withdrawn.  The  cases  treated  by  Flexner 
and  Jobling 's  method  have  now  reached  large  numbers,  both  in 
this  and  foreign  countries  and  the  value  of  the  serum  as  a  therapeutic 
agent  seems  firmly  established. 

The  Serum. — In  America  polyvalent  serum  is  used  almost 
universally.  Horses,  as  for  other  serum  production,  are  the 
animals  employed.  The  cultures  with  which  the  horses  are  im- 
munized must  be  many  containing  representatives  of  the  normal 


37  Wassermann,  Dent,  med.  Woch.,  39,  1907. 

38  Flexner  and  Jobling,  Jour.  Exper.  Med.,  x,  1908. 


530  PATHOGENIC   MICROORGANISMS 

and  para-types,  and  a  considerable  number  of  intermediates,  if  pos- 
sible, to  represent  individuals  from  various  parts  of  what  we  may 
call  the  spectrum  of  intermediate  agglutination  types.  The  choice 
of  cultures  is  perhaps  the  most  important  single  feature  in  serum 
production,  and  those  who  undertake  to  produce  serum  should  be 
constantly  receiving  cultures  from  various  parts  of  the  country, 
isolated  from  cases,  checking  them  up  with  their  serum  product, 
and  adding  them  to  their  immunizing  collection,  if  they  are  not 
represented  by  antibodies  in  the  polyvalent  serum.  It  is  still  a 
question  of  demanding  some  research,  whether  or  not  a  definite 
limitation  of  the  number  of  strains  used  for  immunization  would 
be  of  advantage,  since  the  use  of  too  many  different  strains  may 
keep  down  the  agglutination  value  of  the  serum  of  the  immunized 
horse.  The  cultures  injected  into  the  horse  are  grown  on  agar, 
and  once  washed  in  salt  solution  before  injection.  Various  routines 
for  the  injection  of  horses  have  been  devised,  the  most  useful  method 
at  the  present  time  consisting  of  injecting  on  two  or  three  consecu- 
tive days,  giving  rests  of  seven  or  eight  days,  between  courses  of 
injection.  By  this  method,  antibody  production  may  be  speeded  up. 
It  is  unnecessary  here,  however,  to  go  into  the  details  of  the  actual 
technical  procedures  and  measurements  used  in  the  production  of 
serum.  These  methods  are  constantly  changed  and  can  be  learned 
only  by  taking  part  in  the  process  in  a  well  equipped  producing 
laboratory. 

Serum  must  be  standardized  before  it  can  be  marketed.  This 
has  been  a  very  difficult  matter  and  a  number  of  suggestions  have 
been  made.  Flexner  and  Jobling39  first  attempted  standardization 
by  opsonin  contents.  Complement  fixation  has  been  recommended 
by  some  writers,  but  the  usual  method  at  the  present  time  is  that 
of  agglutination.  As  a  general  rule  Flexner  states  that  the  poly- 
valent sera  are  ready  for  use  when  they  agglutinate  the  normal 
and  para-types  in  dilutions  of  1 :1500  or  1 :2000.  Such  sera  should 
also  agglutinate  intermediate  strains  in  dilutions  of  1 :200  and 
upward. 

Administration  of  Serum. — The  most  important  single  considera- 
tion in  serum  treatment  of  meningitis  is  the  early  recognition  of 
the  case  and  avoidance  of  delay  in  starting  the  specific  treatment. 
Failure  of  serum  treatment  can  probably  in  most  cases  be  referred 

89  Flexner  and  Jobling,  Jour.  Exper.  Med.,  10,  1908. 


MICROCOCCUS    INTRACELLULARLS    MENINGITIDIS          531 

to  delay.  Lumbar  puncture,  therefore,  should  be  done  as  early  as 
the  first  suspicion  is  aroused,  and,  if  meningococci  are  found,  the 
injection  of  serum  should  follow  as  rapidly  as  possible. 

It  is  probably  best,  in  the  long  run,  to  inject  serum  immediately 
upon  obtaining  a  turbid  fluid  in  a  case  in  which  the  clinical  suspicion 
points  strongly  to  epidemic  cerebrospinal  meningitis. 

The  technique  of  serum  injection  consists  in  first  withdrawing 
spinal  fluid  by  tapping  the  canal  with  a  sterile  needle  and  allowing 
the  fluid  to  flow  out,  of  course  without  suction,  holding  a  centrifuge 
tube  directly  over  the  butt  of  the  needle.  The  flow  is  allowed  to 
continue  until  the  drops  begin  to  come  quite  slowly,  that  is,  a  drop 
every  ten  or  twenty  seconds,  and  then  the  serum  is  injected,  either 
by  gravity  or  with  a  syringe  through  the  same  needle.  It  is  im- 
portant that  the  serum  at  body  temperature  shall  enter  the  canal 
very  slowly,  and,  for  this  reason,  the  gravity  method  is  advised.  A 
gravity  arrangement  can  easily  be  constructed  by  attaching  about 
eighteen  inches  of  catheter  tubing,  sterilized,  to  the  end  of  the 
needle  with  a  small  sterile  funnel  at  the  other  end.  The  withdrawal 
of  large  amounts  of  fluid  suddenly  sometimes  causes  trouble,  the 
patient  breathing  rapidly,  and  showing  symptoms  of  threatened 
collapse,  but  this  is  rare,  and  a '  little  judgment  in  withdrawing 
fluid  which  has  been  under  considerable  pressure  too  rapidly  will 
usually  guard  against  accident.  During  the  injection  of  the  serum, 
the  patient  should  be  carefully  watched,  since  occasionally  alarming 
symptoms  may  arise  from  too  rapid  increase  of  internal  pressure. 
The  physician  must  be  on  the  alert  for  such  symptoms  and  imme- 
diately discontinue  the  injection  for  the  time  being.  Flexner40 
advises  10  minutes  for  the  injection  of  the  entire  amount  of  serum 
used. 

The  dosage  of  serum  should,  to  some  extent,  depend  upon  the 
amount  of  fluid  withdrawn,  and  the  amount  injected  should  usually 
be  less  by  several  centimeters  than  the  amount  withdrawn.  The 
average  dose  for  an  adult  should  be  about  30  c.c.,  though  more  may 
be  given  when  large  quantities  of  fluid  have  been  withdrawn,  and 
when  the  case  is  very  carefully  watched  by  an  experienced  man. 
Sophian41  has  recommended  controlling  the  withdrawal  of  spinal 
fluid  and  the  injection  'of  the  serum  by  blood  pressure  measurements. 


40  Flexner,  Bulletin,  Eock.  Inst.  for  Med.  Ees.,  1917. 

n,  Epidemic  Ccrebrospinal  Meningitis,  St.  Louis,  1913,  p.  54. 


532  PATHOGENIC  MICROORGANISMS 

Sudden  drops  of  blood  pressure  in  either  case,  should  lead  to  caution, 
and  perhaps  interruption  of  the  procedure. 

Repetition  of  the  injections  is  as  important  as  the  initial  injec- 
tion, as  far  as  cure  is  concerned.  The  action  of  the  serum  may 
be  compared  somewhat  to  the  action  of  anti-serum  in  a  Pfeiffer 
reaction  in  a  guinea  pig's  peritoneum.  Thus,  probably  some  bac- 
teriolysis and  considerable  stimulation  to  phagocytosis  by  opsonic 
action  may  result.  The  spinal  fluid  shows  changes  in  that  the  num- 
bers of  organisms  are  diminished  and  the  extracellular  ones  dis- 
appear. Purulent  spinal  fluid  may  become  clearer  and  may  even 
become  entirely  free  from  organisms  or  leucocytes.  There  is  prob- 
ably a  certain  amount  of  poison  neutralization  by  the  serum.  Repeti- 
tions of  the  doses,  therefore,  must  be  governed  to  some  extent  by 
the  progress  of  the  case,  clinical  conditions  pointing  to  changes  in 
the  meningeal  inflammation,  and  observation  of  the  spinal  fluid.  One 
injection  a  day  for  three  to  six  days  usually  controls  a  case  that  is 
treated  with  sufficient  promptness. 

In  addition  to  the  intraspinous  administration,  it  is  wise  to  inject 
from  30  to  50  c.c.  intravenously,  preceding  this  by  withdrawal  of 
blood  for  blood  culture,  and  being  governed  as  to  repetition  by 
subsequent  blood  culture  control.' 

Everyone  dealing  with  meningitis  during  an  epidemic  must  re- 
member that  occasionally  meningococcus  septicemia  cases  occur 
which  never  show  meningeal  infection.  We  have  mentioned  these 
in  another  place,  but  believe  that  more  attention  should  be  given 
them,  since  they  are  very  apt  to  be  fatal,  either  without  meningitis, 
or  subsequently  followed  by  a  violent  meningeal  involvement.  Such 
cases  displayed  the  clinical  picture  of  a  general  severe  septic  in- 
fection with  usually  a  profuse  eruption  in  which  petechial  spots 
not  unlike  those  of  typhus  fever  may  cover  the  entire  body.  There 
is  an  irregular  septic  temperature  with  a  high  leucocytosis  and 
sometimes  delirium.  Blood  culture  will  diagnose  these  cases  and 
vigorous  intravenoiis  serum  treatment  would  be  indicated. 

Effects  of  Serum  Treatment. — The  mortality  of  meningitis  in  the 
days  before  serum  was  used  varied  between  60  and  80  per  cent. 
Higher  mortalities  have  been  noted  in  individual  epidemics.  The 
average  for  many  different  parts  of  the  world  fluctuates  about  70 
per  cent.  Since  serum  treatment  was  begun  just  before  the  year 
1906,  a  great  many  statistical  studies  have  been  made  which  are 
of  course  subject  to  great  error,  owing  to  the  fact  that  the  treated 


MICROCOCCUS    INTRACELLULARIS    MENINGITIDIS 


533 


eases  must  have  included  a  great  many  treated  too  late  to  permit 
any  kind  of  treatment  to  be  effective.  Flexner 's  statistics  of  cases 
under  serum  treatment  show  that,  of  1211  cases,  analyzed,  those 
treated  between  the  first  and  third  day  (199)  showed  a  mortality 
of  18.1  per  cent,  those  treated  between  the  fourth  and  seventh  day 
(346)  showed  a  mortality  of  27.2  per  cent,  and  those  treated  later 
than  the  seventh  day  (666)  showed  a  mortality  of  36.5  per  cent. 
The  following  table  taken  from  a  paper  by  Flexner,  published  by 
the  Rockefeller  Institute  as  a  Bulletin  in  1917,  gives  similar  com- 
parative mortality  statistics  reported  by  different  observers. 

COMPARATIVE    MORTALITY  REPORTED  BY  VARIOUS  OBSERVERS  42 


Treatment  Begun. 

Flexner, 
Per  Cent. 

Netter, 
Per  Cent. 

Dopter, 
Per  Cent. 

Christo- 
manos, 
Per  Cent. 

Levy, 
Per  Cent. 

Flack, 
Per  Cent. 

Before  third  dav 

18    1 

7    1 

8  2 

13  0 

13  2 

9   09 

From  fourth  to  seventh  day 

27.2 

11.1 

14.4 

25.9 

20.4 

After  seventh  day  

36.5 

23.5 

24.1 

47.0 

28.6 

50.00 

Altogether,  then,  it  seems  quite 'clear  that  serum  treatment  has 
made  a  tremendous  difference  in  the  mortality  from  this  otherwise 
so  fatal  disease. 


OTHER    GRAM-NEGATIVE    MICROCOCCI   WHICH   MUST    BE 
DIFFERENTIATED  FROM  MENINGOCOCCI 

MICROCOCCI  CATARRHALIS. — This  organism  is  more  particularly 
described  in  a  separate  section  below.  It  is  one  of  the  common 
organisms  which  may  confuse  carrier  examinations  because  of  its 
frequent  presence  in  the  nose  and  throat  of  normal  human  beings. 
Its  fermentation  reactions  are  described  in  the  table  from  Elser  and 
Huntoon43  also  given  below.  The  organisms  are  slightly  larger  than 
meningococci,  grow  readily  on  the  simplest  media,  the  colonies  are 
larger,  thicker,  opaque  and  white,  and  have  a  tendency  to  dryness 
quite  distinct  from  the  dew-drop  like  appearance  of  meningococcus 
colonies,  and  do  not  agglutinate  in  specific  serum.  They  show  a 


42  Flexner,  Bulletin  of  the  Rock.  Inst.  for  Med.  Ees.,  1917, 

43  Elser  and  Huntoon,  Jour.  Med.  Res.,  20,  1909,  371, 


534  PAT  H(Xi  UN  1C   M ICUOOKU AN  ISMS 

tendency  to  spontaneous  agglutination  in  salt  solution  and  in  horse 
serum. 

MICROCOCCUS  FLAVUS. — A  common  inhabitant  of  the  normal  throat 
which  grows  easily  on  simple  media  and  may  be  grown  at  room 
temperature  at  or  below  25°,  temperatures  at  which  the  meningo- 
coccus  ceases  to  grow.  It  is  always  important  to  expose  suspected 
cultures  at  room  temperature  in  the  dark.  A  yellowish  pigment  is 
formed  by  the  cultures,  but  often  does  not  come  out  for  several 
days.  The  very  young  colonies  may  very  closely  resemble  meningo- 
coccus  colonies,  but  are  easily  distinguished  in  sub-cultures,  es- 
pecially when  the  growth  is  forty-eight  or  more  hours  old.  There 
are  a  considerable  number  of  chromogenic  organisms  closely  related 
to  the  Flavus.  Elser  and  Huntoon  describe  three  chief  chromogenic 
groups,  one  of  which  has  a  greenish  gray  or  greenish  yellow  ap- 
pearance by  reflected  light,  with  an  opacity  that  approximates  the 
meningococcus  colony.  The  second  group  is  the  one  most  closely 
resembling  Lingelsheim 's  M.  Flavus. 

Their  third  chromogenic  group  also  makes  a  greenish  yellow 
pigment,  and,  except  for  this,  is  very  similar  to  the  M.  catarrhalis. 
A  curious  fact  has  been  noted  by  Elser  and  Huntoon,  namely,  that 
some  of  their  chromogenic  organisms  were  easily  distinguishable 
from  meningococcus  colonies  at  first  isolation,  but  in  the  course  of 
artificial  cultivation  they  lost  some  of  their  original  characters  and 
their  power  to  produce  pigment,  and  gradually  approximate  the 
appearance  of  meningococcus,  at  least  as  it  appears  in  strains  long 
isolated. 

The  Flavus  group  gives  pernaps  most  difficulty  in  meningococcus 
carrier  examinations,  since  the  young  colonies  of  these  organisms 
may  look  very  much  like  the  young  meningococcus  cultures.  The 
chief  points  of  differentiation,  apart  from  sugar  fermentation,  which 
confirm  them,  are:  The  fact  that  Flavus  colonies  will  grow  out  at 
room  temperature  on  slants  of  simple  media;  that  they  begin  to 
form  pigment  after  forty-eight  hours  or  so,  and  that  they  will 
agglutinate  in  normal  horse  serum  in  dilutions  often  as  high  as  1  to 
50,  and  in  the  meningococcus  sera,  indiscriminately,  often  as  high 
as  1  to  100.  Meningococci  do  not  agglutinate  in  salt  solution  spon- 
taneously, unless  under  the  conditions  mentioned  above  as  noted  by 
Hine,  and  under  the  influence  of  abnormal  acid  or  alkalin  reactions. 
In  all  series  in  which  the  specific  a  i>^luti  nation  test  is  used  for  1he 
determination  of  a  meningococcus,  therefore,  normal  hoi-sc  scrum 


MICROCOCCUS    INTRACELLUDARIS    MENINGITIDIS 


535 


tubes  should  be  set  up  in  dilutions  ranging  up  to  1  to  50  at  least, 
in  order  to  exclude  organisms  of  the  Mavus  type. 

MICROCOCCUS  PHARYNQIS  Siccus. — This  organism  described  by 
Lingelsheim44  is  a  Gram-negative  diplococcus  often  found  in  the 
normal  pharynx,  and  is  recognized  by  its  dry,  creiiated  colonies 
on  simple  media.  According  to  Elser  and  lluntoon,  it  sediments 
spontaneously  in  salt  solution  and  this,  together  with  the  fact  that 
the  colonies  are  formed  in  a  way  almost  impossible  to  break  up, 
makes  it  easy,  according  to  these  observers,  to  distinguish  it  from 
the  meningococcus.  It  is  a  little  more  difficult  to  distinguish  from 
M.  Catarrhalis;  but  can  be  easily  separated  from  this  organism  by 
means  of  the  fermentation  test. 

DIPLOCOCCUS  CRASSUS. — This  is  the  organism  that  Kutscher45 
described  as  probably  identical  with  the  so-called  "Jaeger"  variety 
of  meningococcus.  According  to  Kutscher  and  von  Lingelsheim, 
this  organism  has  a  tendency  to  wander  from  the  normal  pharynx 
into  the  central  nervous  system  in  cases  of  meningitis  of  other 
origin.  Lingelsheim  claims  to  have  found  it  in  the  fluids  of  traumatic 
meningitis  and  tuberculous  meningitis.  It  has  the  peculiarity  that 
the  cultures  are  said  to  be  composed  of  Gram-negative  and  Gram- 
positive  organisms  some  of  the  cocci  retaining  the  Gram-stain.  Ac- 
cording to  Von  Lingelsheim,  the  colonies  are  smaller  and  more 
compact  than  meningococcus  colonies,  and  it  will  grow  at  room 
temperature. 

FERMENTATION    REACTIONS    OF     GRAM     NEGATIVE     DIPLOCOCCI 


Strains  Tested. 

Strains 

Dex- 
trose. 

Mal- 
tose. 

Levu- 
lose. 

Sacch- 
arose. 

Lac- 
tose. 

Gal- 
actose. 

Meningococcus  

200 

-f- 

+ 

0 

0 

0 

0 

Pseudomeningococcus  
Gonococcus  

6 
15 

+ 
+ 

+ 
0 

0 

0 

0 
0 

0 
0 

0 
0 

Micrococcus  catarrhalis  
Micrococcus  pharyngis  siccus..  . 
Chromogenic  group  I 

64 

2 
28 

0 

+ 

+ 

0 

+ 
+ 

0 

+ 
+ 

0 

+ 

+ 

0 
0 
0 

0 
0 
0 

Chromogenic  group  II 

11 

4. 

+ 

-f 

0 

0 

0 

Chromogenic  group  III.  .  .  
Jaeger  meningococcus,  Krai  .... 
Diplococcas  crassus  Krai  

9 
1 
1 

+ 
+ 
+ 

+ 
+ 

+ 

0 

+ 
+ 

0 

+ 
+ 

0 

+ 
+ 

0 

+ 

+ 

Table  taken  from  Elser  and  Huntoon,  loc.  cit. 


44  Lingelsheim,  Klin.  Jahrb.,   15,  1906. 

45  Kutscher,  Kolle  and  Wassermann,  Vol.  4,  Second  Edition,  p.  603. 


536  PATHOGENIC  MICROORGANISMS 

DIPLOCOCCUS  Mucosus. — A  form  of  Gram-negative  diplococcus, 
the  description  of  which  we  take  from  Elser  and  Huntoon.  Its 
colonies  may  resemble  meningococcus  colonies  on  ascitic  agar.  They 
are  said  to  differ  from  the  meningococcus  colonies  by  being  more 
mucoid,  resembling  the  colonies  of  the  B.  capsnlatus  mucosus.  The 
colonies  have  a  tendency  to  confluence  and  the  above  writers  say 
that  the  luxuriance  of  its  growth  on  serum  free  media  helps  to  tell 
it  from  the  meningococcus.  It  also  grows  at  room  temperature,  and 
shows  capsules  with  capsule  stains. 


EPIDEMIOLOGICAL    PROBLEMS    IN   MENINGITIS 

Although  sporadic  cases  of  meningitis  may  occur  in  a  community 
for  a  considerable  number  of  years  after  an  epidemic  is  over,  and 
the  disease  may,  therefore,  be  regarded  as,  to  some  extent,  endemic 
in  all  crowded  cities,  it  is  chiefly  important  for  its  epidemic  occur- 
rence. In  1905  a  recognizably  described  epidemic  occurred  in 
Switzerland.  Since  that  time  epidemics  have  been  reasonably  fre- 
quent, especially  at  times  of  war,  when  they  appeared  among  armies 
in  barracks  and  mobilization  camps.  During  the  many  continental 
campaigns  in  the  time  of  Napoleon,  outbreaks  occurred  in  the 
various  armies,  and  secondary  epidemics  among  the  civilian  popula- 
tion in  many  cities  followed  in  the  train  of  these.  In  America,  a 
number  of  limited  epidemics  occurred  in  the  States  along  the  Eastern 
sea  board  during  the  early  half  of  the  19th  century,  and  in  these 
civilian  epidemics  the  disease  particularly  selected  children  and 
young  adults.  Extensive  civilian  epidemics  occurred  in  different 
parts  of  the  world  in  the  early  years  of  the  20th  century.  In  1903 
the  disease  appeared  in  East  Prussia  and  spread  to  other  parts  of 
Germany  from  there.  In  1904  and  1905  it  appeared  in  New  York 
City,  and  the  adjacent  country,  on  an  extensive  scale  causing  the 
death  of  3,000  people  and  altogether  about  7,000  cases  in  New  York 
City  alone.  In  the  summer  following  its  appearance  in  New  York, 
it  extended  to  Canada,  and  in  the  years  since  then,  small  outbreaks 
and  sporadic  cases  have  appeared  with  gradually  decreasing  fre- 
quency all  through  the  more  thickly  populated  parts  of  North 
America. 

During  the  late  war,  there  was  little  meningitis  among  the 
European  armies  until  an  extensive  outbreak  occurred  among  the 


MICROCOCCUS    INTRACELLULARIS    MENINGIT1DIS         ( 537 

Canadian  troops  on  Salisbury  Plains.  The  increase  of  cases  among 
these  troops  took  place  in  February,  1915,  and  after  this  time  the 
disease  began  to  appear  in  the  overseas  expeditionary  troops,  al- 
though among  these  no  extensive  epidemic  occurred  at  any  time. 
Among  American  troops  the  disease  was  most  prevalent  in  1917 
and  1918  among  the  troops  gathered  in  the  cantonments  in  the 
United  States.  According  to  the  epidemiological  studies  of  Vaughan  ( 
and  Palmer4'5  in  the  camps  in  1918,  meningitis  showed  "of  all  dis- 
eases, the  greatest  excess  over  the  disease  in  civilian  communities.'* 
Vaughan  estimates  that  meningitis  was  forty-times  as  frequent  in 
the  Army  as  in  civilian  life.  The  highest  morbidity  occurred  at 
Camp  Jackson  where  it  reached  a  rate  of  25.1  per  thousand,  and  a 
death  rate  of  7.05.  Next  to  pneumonia,  it  was  the  most  serious  disease 
occurring  in  the  camps.  In  the  Surgeon  General's  report  for  1918, 
the  disease  stood  fifth  as  a  cause  of  death  for  enlisted  men  in  the 
United  States  and  Europe,  with  a  case  mortality  of  34.8  per  cent. 

During  the  Army  epidemics  there  was  a  definite  racial  difference 
in  that,  according  to  Surgeon  General  Ireland's  report,  the  admission 
rate  for  colored  troops  in  the  United  States  was  2.44,  whereas,  it 
was  only  1.2  for  whites,  and  the  death  rate  for  colored  troops  was 
0.98  against  0.41  for  whites. 

As  to  seasonal  prevalence,  meningitis  usually  develops  in  the 
late  autumn  and  winter  months,  the  largest  case  rates  being  coin- 
cident with  the  cold  and  wet  weather,  when  a  basic  catarrhal  inflam- 
mation of  the  upper  respiratory  tract,  creates  favorable  conditions 
for  the  lodgment  of  organisms  and  for  the  general  distribution  of 
saliva  by  coughing,  sneezing  and  spitting.  During  the  war,  the 
highest  admission  rates  in  the  United  States  usually  fell  into  the 
months  of  November,  December  and  January,  which  is  the  time  of 
the  highest  case  rate  for  most  respiratory  epidemics.  Yet  cases  will 
usually  trail  along  through  the  hot  weather. 

Meningitis  epidemics,  therefore,  will  occur  chiefly  in  the  tem- 
porate  zones  during  the  winter  months  at  times  when,  during  the 
prevalence  of  generalized  respiratory  disease,  large  numbers  of 
people  are  crowded  in  close  quarters,  under  conditions  which  render 
attention  to  hygiene  and  sanitation  difficult.  The  reasons  for  this 
will  become  apparent  as  we  study  the  manner  of  transmission. 

The  meningocoecus  does  not  survive  easily  outside  the  body,  and 


46  Vauglian  and  Palmer,  Jour.  Lab.  and  Clin.  Med.,  4,  1919,  647. 


538  PATHOGENIC  MICROORGANISMS 

rapidly  dies  out  in  dust,  or  even  in  sputum,  under  conditions  of 
low  temperature,  deficient  moisture  and  competition  with  other 
microorganisms.  As  far  as  we  know,  it  is  not  carried  by  any  of 
the  domestic  animals  and,  therefore,  the  origin  of  infection  lies  in 
the  secretions  of  cases  and  of  carriers.  The  microorganisms  are 
found  in  the  noses  and  throats  of  the  sick,  sometimes  in  the  secre- 
tions of  the  eye  where  a  meningococcus  conjunctivitis  may  exist. 
With  the  secretions  of  these  mucous  membranes  it  reaches  the  outer 
world.  The  meningococci  may  be  present  in  cases  for  a  long  time 
after  convalescence,  and,  as  we  know,  they  are  present  in  a  consider- 
able percentage  of  people  who  have  never  had  meningitis,  with  whose 
secretions  the  organisms  may  be  constantly  transferred  to  contacts. 
Transmission  probably  occurs  by  close  contact  between  carrier  or  case 
and  new  host,  through  the  nasopharynx,  where  the  organisms  lodge  and 
multiply.  From  this  lodgment  they  pass  into  the  meninges,  either 
directly  along  the  lymphatic  channels  to  the  base  of  the  skull,  or 
perhaps  indirectly  by  way  of  the  general  circulation.  The  former 
route  is  the  one  favored  by  most  observers.  However,  during  the 
early  periods  of  the  war,  a  few  cases  were  reported  by  British 
clinicians,  in  which  blood  culture  was  positive  before  symptoms  of 
meningitis  had  developed,  and  during  these  army  epidemics,  we, 
as  well  as  others,  saw  occasional  cases  of  general  meningococcus 
septicemia  which  died  without  ever  developing  meningitis.  In- 
cidentally, it  may  be  stated  that  these  cases  develop  a  generalized 
rash  which,  in  some  of  its  stages,  is  not  unlike  that  of  typhus  fever. 
The  writer  recalls  a  case  in  which  he  made  a  probable  diagnosis  of 
typhus  fever  which  in  the  light  of  subsequent  experience  seems  to 
him  to  have  possibly  been  a  case  of  meningococcus  septicemia. 
Positive  blood  culture  in  such  cases  will  differentiate  between  the 
two  diseases.  Herrick47  studied  this  phase  of  the  problem  at  Camp 
Jackson  in  1918  with  great  care,  and  came  to  the  conclusion  that 
in  50  per  cent  of  the  cases  early  blood  culture  will  reveal  general 
infection  before  clinical  evidences  of  meningeal  invasion  are  ap- 
parent. This  observation  is  of  the  greatest  importance,  indicating 
the  desirability  of  early  blood  culture  work  for  doubtful  diagnosis, 
and  also  throwing  light  upon  the  wisdom  of  intravenous  serum 
therapy  combined  with  the  intraspinous  injections. 

Infection   of  a  healthy  individual   from   a  case  is  of  very  rare 


41  llerrick,  Arch.  Inter.  Med.,  21,  1918,  541. 


MICROCOCCUS    INTRACELLULARIS    MENINGITIDIS  539 

occurrence,  and  since  there  are  in  every  epidemic  a  very  much  larger 
number  of  carriers  than  of  cases,  the  carrier  is  the  chief  epi- 
demiological problem.  As  far  as  infection  of  new  individuals  from 
patients  is  concerned,  experience  during  the  New  York  epidemic 
showed  only  two  or  three  cases  of  infection  of  doctors  and  nurses, 
although  the  hospitals  in  the  city  were  constantly  handling  consider- 
able numbers  of  the  sick.  In  our  epidemiological  experience  with 
the  army,  the  actual  tracing  of  one  case  to  a  preceding  one  was 
also  relatively  very  rare.  This  does  not  mean  that  the  greatest 
precautions  should  not  be  taken  to  prevent  such  transmission  in 
hospitals  and  sick  room.  But  the  epidemiological  emphasis  lies  with 
the  carrier. 

This  rareness  of  transmission  from  cases  to  the  healthy  is  in 
our  opinion  due  to  the  peculiar  conditions  of  susceptibility  that  pre- 
vail in  relation  to  meningitis,  and  the  fact  that  the  number  of 
people  with  whom  the  sick  come  in  contact  is  relatively  small. 

The  susceptibility  of  man  to  meningitis  is  a  curious  one,  different 
in  some  aspects  from  susceptibility  relations  to  almost  all  other 
infections,  except  perhaps  poliomyelitis.  In  the  general  population 
there  seems  to  be  a  great  variability  in  individual  susceptibility  to 
infection  with  the  meningococcus,  a  variation  which  can  be  traced 
to  no  determinable  cause.  Unlike  pneumonia,  temporary  fluctua- 
tions in  well  being,  produced  by  respiratory  disease,  malnutrition, 
exposure  to  cold,  etc.,  do  not  seem  to  play  a  determining  role.  The 
disease  indiscriminately  picks  out  individuals  here  and  there,  some 
of  them  in  the  most  robust  health,  strong  and  hardy,  while  sparing 
associates  who  may  be  feeble  and  run  down.  It  is  obvious  that  some 
individuals  are  normally  resistant  and  will  not  come  down,  in  spite 
of  considerable  exposure,  while  others  are  delicately  susceptible. 
The  difference  may  possibly  have  been  produced  fortuitously  by  the 
fact  that  some  individuals  may  have  been  carriers  at  one  time  or 
another,  and  have  become,  thereby,  spontaneously  immunized.  It 
is  difficult  to  get  at  this  question  experimentally,  and  there  are  no 
serum  or  other  reactions  which  we  can  apply  at  the  present  time, 
by  which  we  can  discriminate  between  the  susceptible  and  the  non- 
susceptible  of  a  community.  There  is  no  available  method,  more- 
over, by  which  we  can  distinguish  between  virulent  and  non- virulent 
strains  of  meningococci. 

The  Carrier  Problem. — As  stated  above,  the  meningococcus  car- 
rier probably  is  the  source  of  infection  in  most  of  the  cases  that 


540  PATHOGENIC   MICROORGANISMS 

develop  during  an  epidemic.  Earlier  carrier  work  has  lost  value 
to  a  considerable  extent,  owing  to  the  fact  that  the  criteria  of 
meningococcus  identification  of  which  we  are  now  more  thoroughly 
informed  were  neglected  in  these  early  studies.  The  flora  of  the 
nose  and  throat  contains  many  Gram-negative  diplococci,  mentioned 
above  under  the  heading  of  identification,  some  of  which  were  mis- 
taken in  this  earlier  work  for  true  meningococci.  Micrococcus 
Catarrhalis,  Micrococcus  Flavus,  and  a  number  of  other  similar 
microorganisms  probably  represent  a  definite  percentage  of  the 
earlier  statistics.  The  criteria  of  meningococcus  determination  have 
been  discussed  in  a  special  section  above,  and  these,  in  general,  were 
applied  in  the  extensive  meningococcus  carrier  work  which  was  done 
during  the  years  of  the  war,  especially  by  British  and  American 
bacteriologists.  The  studies  of  Bassett-Smith,48  Gordon,49  Mathers 
and  Herrold,50  show  that  in  the  American  camps  under  conditions 
of  ordinary  life  and  weather,  there  may  be  anywhere  from  two  to 
five  per  cent  of  meningococcus  carriers.  Mathers  and  Herrold  at 
the  Great  Lakes  Naval  Training  Station  examined  over  15,000  men, 
finding  over  4  per  cent  to  be  carriers  and  between  1  and  2  per  cent 
to  be  chronic  carriers.  Their  work  also  showed  that  the  carrier 
rate  is  higher  among  those  taking  care  of  cases,  and  that  over 
38  per  cent  of  those  recovering  from  the  disease  may  remain  carriers 
during  convalescence  for  variable  periods.  Contacts  showed  a 
carrier  rate  of  36.7  per  cent  during  a  period  in  which  the  general 
carrier  rate  in  the  camps  (15,000  men  examined)  was  slightly  over 
4  per  cent.  The  hospital  Corps  showed  a  carrier  rate  of  13.5  per 
cent.  It  is  natural  that  there  have  been  many  endeavors  to  establish 
relationship  between  new  cases  and  contact  with  carriers.  This  line 
of  investigation  has  not  been  conclusive,  owing  to  the  great  difficul- 
ties incident  to  such  investigation.  The  transmission  of  respiratory 
organisms  may  take  place  during  a  very  brief  contact,  in  conversa- 
tion, close  association  in  barracks,  moving  picture  shows,  public 
conveyances,  sleeping  quarters,  etc.,  and  the  innumerable  associa- 
tions of  this  kind  established  by  each  man  in  the  course  of  a  day, 
makes  it  almost  impossible  to  trace  them  with  accuracy.  Among 
the  most  interesting  studies  made  in  this  connection  are  those  of 


48  Bassett-Smith,  Lancet,  194,  1918,  290. 

"Gordon,  Med.  Res.  Com.,  Spec.  Rep.  Ser.,  No.  3,  London,  1917. 

50  Mathers  and  Herrold,  Jour.  Infect.  Dis.,  22,  1918,  523. 


MICROCOCCUS    INTRACELLULARIS    MENINGITIDIS 


541 


Glover,51  who  swabbed  the  throats  of  a  considerable  number  of  men 
in  overcrowded  barrack  rooms,  in  the  course  of  sanitary  supervision 
during  which  the  spacing  between  beds  was  among  the  many  pre- 
cautionary measures  taken.  It  will  be  seen  here  that  sanitary 
measures,  including  the  spacing  out  in  sleeping  quarters,  brought  about 
a  very  considerable  drop  in  the  carrier  rate,  'with  coincident  diminu- 
tion of  cases  of  meningitis.  Meleney  and  Ray52  traced  fourteen  out 

EFFECTS    OF    "SPACING   OUT"    ON    "SEVERELY    OVER  CROWDED" 

BARRACK-ROOMS  * 


Unit. 

Date  of 
First 
Swabbing. 

Percentage 
Carrier 
Rate  before 
Spacing. 
Out. 

Period 
Spaced 
Out 
Approxi- 
mately. 

Date  of 
Second 
Swabbing. 

Percentage 
Carrier 
Rate  after 
Spacing 
Out. 

No  1 

Sept  29 

22.0 

8  weeks 

Dec.    6 

2.0 

No.  2  

Oct.      2 

28.0 

6  weeks 

Nov.  23 

7.0 

One  room  of  No.  2  
No  4 

Oct.     2 
Oct    26 

38.5 
28  0 

6  weeks 
5  weeks 

Nov.  23 
Nov.  30 

4.5 
4.5 

*Glover's  Table 

of  twenty-four  cases  which  occurred  in  an  American  camp  to  contact 
with  carriers,  and  found  parallelism  between  the  incidence  of  cases 
and  the  rise  of  the  carrier  rate.  These  examples,  however,  are  excep- 
tional and  it  is  relatively  rare  that  a  definite  relationship  of  this 
kind  can  be  established.  It  is  a  fact  that  carrier  rates  are  high 
in  such  camps  during  the  cold  months,  and  in  connection  with  the 
general  spread  of  respiratory  disease,  and  that,  at  such  times,  the 
incidence  of  the  disease  increases,  and  it  is  absolutely  logical  to 
assume  that  the  new  cases  arise  by  contact  with  the  carriers.  It  is 
of  importance,  however,  to  recognize  that  the  tracing  of  the  case 
to  the  individual  from  whom  he  has  been  infected,  at  times  when 
high  carrier  rates  exist,  is  not  often  possible,  and  comprehension 
of  this  must  considerably  influence  the  measures  instituted  for  the 
control  of  the  epidemic. 

It  is  our  belief  that  the  extensive  carrier  examinations  made 
during  epidemics  and  the  wholesale  isolation  of  carriers,  were  rela- 
tively ineffective  during  the  war,  and  that  it  is  far  better  to  bend 
all  one's  energies  upon  a  general  improvement  of  the  respiratory 


51  Glover,  Jour.  Hyg.,  17,  1918,  367. 

52  Meleney  and  Kay,  Jour.  Inf.  Dis.,  23,  1918,  317. 


542  PATHOGENIC  MICROORGANISMS 

sickrate  with  the  reduction  of  carriers,  focusing  the  carrier  ex- 
aminations upon  the  small  epidemiologically  determined  intimate 
group  from  which  the  case  has  come,  rather  than  making  wholesale 
carrier  examinations  of  carriers  of  meningococcus  through  whole 
regiments  and  divisions. 

Carrier  Determination. — The  bacteriological  analysis  of  a  carrier 
is  not  a  simple  procedure,  and  implies  the  proper  control  of  a  great 
many  conditions  which  necessitate  special  description. 

To  obtain  the  material  properly  the  swab  must  be  taken  from 
high  up  in  the  pharynx,  behind  the  soft  palate.  A  general  swabbing 
of  the  pharynx  and  throat  is  not  sufficient.  The  best  swabs  for  this 
purpose  are  made  by  the  West  tube  method,  as  follows:  A  cotton 
swab  is  fixed  on  the  end  of  a  copper  wire,  about  eighteen  centimeters 
long,  and  this  is  inserted  in  a  glass  tube,  bent  upward  at  the  swab 
end,  in  such  a  way  as  to  permit  passage  upward  behind  the  soft 
palate.  The  swab  is  placed  into  the  tube,  both  ends  plugged  with 
cotton,  and  is  so  sterilized.  For  large  scale  work  it  is  sufficient 
to  take  copper  wire  swabs,  sterilize  them  in  a  box,  and  bend  them 
up  carefully  with  the  finger,  being  careful  not  to  touch  the  cotton, 
just  before  use.  Swabbing  through  the  nose  has  also  been  practiced, 
but  we  do  not  believe  that  it  is  as  efficient  as  the  method  described 
above.  The  swab  must  be  taken  with  the  patient  facing  the  light. 
A  tongue  depressor  is  used,  and  the  swab  inserted  so  as  to  pass 
behind  the  soft  palate.  The  copper  wire  is  then  thrust  forward 
so  that  the  swab  emerges  from  the  tube  and  touches  the  posterior 
and  upper  pharyngeal  wall.  Slow  motion  to  and  fro  brings  the 
cotton  in  contact  with  the  sides  of  the  upper  pharynx.  The  swab 
is  then  immediately  passed  over  the  surface  of  the  plate  medium. 
It  is  best  not  to  carry  the  inoculated  swab  back  to  the  laboratory, 
but  to  plate  it  directly  upon  removing  the  material  from  the  patient. 

The  media  employed  are  various,  but  for  ordinary  use  a  glucose- 
hormone-agar,  PH  of  7.4  with  addition  of  1-10  defibrinated  or 
hemolyzed  human  or  rabbit's  blood,  is  best.  The  plates  thus 
inoculated  should  be  kept  warm  and  immediately  taken  to  the 
laboratory  where  they  are  incubated.  The  British  prefer  trypagar 
to  the  hormone  agar  as  the  basic  medium  for  such  work. 

After  eighteen  to  twenty-four  hours  incubation,  the  plates  are 
examined  and  the  colonies  suspected  of  being  meningbcocci  are 
fished.  This  is  not  a  matter  which  can  be  taught  by  book.  The 
colonies  are  of  small,  rounded  appearance,  the  recognition  of  which 


MICROCOCCUS    INTRACELLULARIS    MENINGITIDIS  543 

is  a  matter  of  judgment.  Every  bacteriologist  confronted  with  the 
problem  should  immediately  plant  plates  of  the  medium  which  he 
is  going  to  use,  with  spinal  fluid  or  with  cultures  recently  isolated, 
and  familiarize  bimself  with  the  colonies  on  this  medium,  at  various 
stages  of  growth.  In  spite  of  our  not  inconsiderable  experience,  we 
would  do  this  ourselves.  Plates  that  are  too  thickly  covered  with 
'colonies  are  of  no  use. 

On  blood  medium,  the  true  meningococcus  colonies  do  not  produce 
any  change  in  the  blood.  They  are  slightly  translucent  and  look 
somewhat  stringy.  They  are  homologous  and  slightly  glistening. 
They  are  practically  indistinguishable  from  young  colonies  of  M. 
Flavus  which  is  also  a  Gram-negative  diplococcus,  but  which  is  easily 
distinguished  subsequently  by  the  fact  that  it  will  grow  at  and 
below  25°  C.,  produces  a  yellow  pigment  on  further  cultivation,  and 
has  a  tendency  to  agglutinate  spontaneously  in  normal  horse  serum. 

Having  ringed  the  suspicious  colonies,  some  of  them  are  now 
picked  and  stained  by  Gram.  Our  habit  is  to  take  up  part  of  a 
colony  which  we  strongly  suspect  of  being  meningococcus,  plant  part 
of  it  immediately,  and  from  the  rest  make  the  Gram  stain,  since 
plating  after  taking  material  for  Gram  stain  may  increase  the 
chances  for  contamination.  This  point,  however,  is  not  of  very  great 
importance. 

The  suspicious  colonies  are  now  planted  upon  blood  agar  slants, 
the  medium  being  made  up  as  for  the  plates.  These  slants  should 
not  be  used  directly  from  the  ice-box,  but  should  be  warmed.  Dried 
media  must  not  be  used. 

If  two  tubes  can  be  inoculated,  one  should  be  kept  at  room 
temperature.  Growth  in  the  incubator  for  about  twelve  hours  gives 
sufficient  growth  for  further  identification. 

Gram  stains  are  now  made  from  the  tubes. 

If  the  morphological  and  staining  properties  are  proper,  agglu- 
tination is  carried  out. 

Diagnostic  Agglutination. — The  organisms  are  emulsified  in 
isotonic  salt  solution.  Agglutination  may  be  done  against  type  sera, 
or  against  polyvalent  serum.  When  large  numbers  of  cases  are 
examined,  as  in  times  of  epidemic,  it  is  best  first  to  agglutinate 
in  polyvalent  serum,  preferably  one  in  which  the  agglutinin  titre 
for  a  great  many  different  meningococcus  strains  has  been  con- 
trolled. As  a  general  rule,  the  polyvalent  sera  used  in  this  country 
will  show  specific  agglutinations  for  practically  all  meningococcus 


544  PATHOGENIC   MICROORGANISMS 

strains  in  dilutions  of  1  to  100.  For  this  reason,  1  to  100  was  the 
dilution  adopted  for  such  work  in  the  American  Army  laboratories. 

One-half  c.c.  of  the  bacterial  emulsion  is  mixed  with  0.5  c.c.  of 
the  polyvalent  serum.  A  control  of  a  similar  amount  of  the  culture 
suspension  in  1  to  50  normal  horse  serum  must  always  be  made  to 
guard  against  spontaneous  agglutination.  It  is  always  well  also, 
to  run  a  tube  with  a  known  meningococcus. 

Since  meningococci  show  a  certain  amount  of  resistance  to  ag- 
glutination, Gordon  has  recommended  the  method  in  general  use 
during  the  war,  that  is,  placing  the  tubes  in  a  water  bath  at  50° 
for  twelve  to  eighteen  hours.  Evaporation  must  be  guarded  against. 

Olitsky  has  recommended  saving  time  by  growing  the  organisms 
in  normal  horse  serum  broth  directly  from  the  colonies  on  the  plate, 
discarding  all  those  that  grow  in  a  granular  form. 

We,  ourselves,  have  used  a  method  that  we  have  not  published 
because  we  have  not  had  a  chance  to  use  it  on  material  on  a  large 
scale,  which  depends  upon  the  thread  reaction.  Dilutions  of  poly- 
valent sera  are  made  in  broth  tubes,  so  that  the  final  concentration 
is  1  to  100.  The  colony  is  directly  inoculated  into  this,  and  in  the 
case  of  true  meningococci  grow  in  granular  form.  A  similar 
inoculation  is  made  in  control  tubes  of  1  to  50  normal  horse  serum. 
These  procedures  sometimes  save  time. 

Nicolle  in  France  makes  his  diagnosis  by  another  method,  in  that 
he  uses,  instead  of  a  dilution  of  serum,  the  serum  in  concentrated 
form,  checking  it  up  with  the  bacterial  suspension,  and  noting  the 
speed  of  agglutination.  In  such  concentrations  he  often  gets  rapid 
agglutination  of  true  meningococci  in  the  concentrated  serum. 

After  the  preliminary  identification  has  been  made,  typing  of  the 
meningocococus  may  be  desirable  by  agglutination  against  type 
serum. 

Carriers  occasionally  will  develop  meningitis  some  time  after 
they  have  been  recognized  as  carriers,  showing  that  the  organisms 
may  remain  in  the  nasopharynx  for  some  time,  without  penetrating, 
insusceptible  individuals.  We  know  of  a  number  of  cases  in  which 
this  seems  to  have  occurred.  Gordon  mentions  a  number  of  cases 
in  which  "meningismus"  developed  among  carriers,  namely,  carriers 
complained  of  severe  headache,  pains  in  the  back  of  the  neck,  slight 
fever  up  to  102°,  and  slight  Kernig.  One  case  he  mentions  had 
been  in  contact  for  a  few  hours  with  a  case  of  cerebro-spinal 
meningitis,  which  died  within  twenty-four  hours.  It  was  swabbed 


M1CROCOCCUS    INTRACELLULARIS    MENINGITIDIS  545 

and  found  to  be  negative.  A  month  later,  he  went  to  a  military 
hospital  with  the  symptoms  above  enumerated,  and  the  swab  from 
his  nasopharynx  revealed  meningococcus,  but  curiously  not  of  the 
same  type  as  that  of  the  case  with  which  he  had  been  in  contact. 
He  mentions  other  similar  cases. 

An  interesting  point  comes  up  in  regard  to  whether  or  not  the 
presence  of  meningococcus  in  the  nasopharynx  leads,  in  itself,  to  a 
catarrhal  inflammation.  Fliigge  in  the  early  days  of  meningococcus 
carrier  investigation  believed  that  the  carrier  state  was  usually 
associated  with  local  inflammations.  Gordon,  however,  finds  that, 
in  general,  there  was  no  nasopharyngeal  catarrh  associated  with  the 
carrier  state.  But  he  also  finds  that_  cases  with  tonsillar  or  pharyn- 
geal  inflammations  were  much  more  difficult  to  free  from  meningo- 
coccus than  others.  The  same  he  says  is  true  of  convalescents,  a 
point  which  indicates  the  importance  of  bringing  the  mucous  mem- 
branes to  normal  in  connection  with  the  cure  of  carriers. 

The  question  of  how  we  are  to  deal  with  meningococcus  carriers 
in  times  of  epidemic  is  a  difficult  one.  Local  treatment  .of  the  nose 
and  throat  has  been  tried  with  antimeningococcus  serum,  with 
astringent  solutions,  and  various  disinfectants,  without  encouraging 
result.  Sprays  of  Dichloramine  T  and  other  chlorin  preparations 
have  been  tried,  also,  in  our  opinion,  without  marked  success.  Dur- 
ing the  war  the  British  constructed  rooms  of  about  one  thousand 
cubic  feet  capacity,  along  the  sides  of  which  steam  pipes  were 
placed  at  about  the  height  of  a  man's  waist,  and  jets  were  fitted 
to  them  in  such  a  way  that  a  spray  of  steam  could  be  ejected.  These 
sprays  were  connected  with  bottles  containing  1  to  2  per  cent 
chloramine,  or  0.5  per  cent  zinc  sulphate.  The  carriers  were  put 
into  these  inhaling  rooms  for  from  fifteen  to  twenty  minutes  a  day, 
during  which  they  inhaled  the  medicated  spray  through  their  nos- 
trils. By  this  method,  they  claimed  to  clear  up  all  but  the  most 
resistant  cases  of  so-called  pure  meningococcus  carriers.  In  general, 
it  may  be  said  that  cases  in  which  only  a  few  meningococcus  colonies 
form  on  the  plates,  clear  up  rather  readily,  and  that  the  others 
in  which  the  cultures  are  almost  pure  are  extremely  resistant  to  any 
kind  of  treatment.  Our  own  impression  from  some  experience  with 
the  various  methods  would  lead  us  to  conclude  that  the  best  treat- 
ment for  a  carrier  would  be  careful  attention  to  the  nasopharynx, 
with  an  attempt  to  bring  it  back  to  normal  as  far  as  the  condition 
of  the  mucous  membrane  is  concerned,  correction  of  tonsillar,  adenoid, 


546  PATHOGENIC   MICROORGANISMS 

or  septum  defects,  cessation  from  smoking  or  other  habits  that 
irritate  the  mucous  membrane,  and  outdoor  life,  especially  in  the 
sunlight,  with  sea  baths  if  available.  Specific  antiseptic  treatment 
in  general  seems  to  us  to  have  been  a  failure  as  far  as  the  handling 
of  large  numbers  of  men  is  concerned. 

The  virulence  of  meningococci  is  a  matter  that  is  very  difficult 
to  determine  because  of  our  inability  to  produce  invasive  infections 
with  regularity  in  any  known  laboratory  animal.  So  far,  extensive 
attempts  to  determine  the  virulence  of  standardized  injections  into 
mice  have  not  succeeded.  Death  in  most  laboratory  animals  is  due 
to  the  toxic  effects  and  not  by  invasion.  This  is  a  very  unfortunate 
circumstance,  inasmuch  as  our  failure  to  be  able  to  distinguish 
between  virulent  and  non-virulent  strains  makes  it  impossible  for 
us  to  tell  a  dangerous  carrier  from  one  who  is  relatively  harmless, 
as  we  can  in  the  case  of  diphtheria  carriers.  All  we  can  do  at  the 
present  time  is  to  regard  as  dangerous  any.  carrier  whose  meningo- 
coccus  agglutinates  in  a  polyvalent  serum.  Those  with  strains  which 
neither  agglutinate  nor  absorb  with  the  polyvalent  serum  at  our 
disposal,  if  culturally  they  seem  to  be  true  meningococci,  must  be 
regarded  as  suspicious. 


CHAPTER  XXVII 


DIPLOCOCCUS     GONORRHCE^E      (GONOCOCCUS),     MICROCOCCUS 
CATARRHALIS,     AND     OTHER    GRAM-NEGATIVE     COCCI 

DIPLOCOCCUS    GONORRHOEA 

NEissER,1  in  1879,  described  diplococci  which  he  had  found  regularly 
in  the  purulent  secretions  of  acute  cases  of  urethritis  and  vaginitis  and 
in  the  acute  conjunctivitis  of  the  new-born.  His  researches  were  purely 
morphological,  as  were  the  numerous  confirm- 
atory investigations  which  rapidly  followed  his 
announcement. 

Cultivation  of  this  diplococcus,  now  usually 
spoken  of  as  gonococcus,  was  not  definitely  suc- 
cessful until  1885,  when  Bumm  2  obtained  growth 
upon  tubes  of  coagulated  human  blood  serum. 
Bumm  was  not  only  able  to  keep  the  organisms 
alive  by  transplantation  in  pure  culture,  but 
produced  the  disease  by  inoculation  of  his  cul- 
tures upon  the  healthy  urethra. 

Morphology  and  Staining. — The  gonococcus 
is  usually  seen  in  the  diplococcus  form,  the  pairs 
being  characteristically  flattened  along  the  sur- 
faces facing  each  other.  This  gives  the  cocci  a 
peculiar  coffee-bean  or  biscuit  shape.  The  size 
of  the  diploforms  is  about  1.6  micra  in  the  long 
diameter,  about  0.8  micron  in  width.  Stained 
directly  in  gonorrheal  pus  from  acute  cases,  the 
microorganisms  are  found  both  intra-  and  extra- 

cellularly,  a  large  number  of  them  crowded  characteristically  within 
the  leucocytes.  They  are  never  found  within  the  nucleus.  The 
phagocytosis  which  produces  this  picture  has  beeji  shown  by  Scholtz  3 


? 


FIG.  57. — GONORRHEAL 
Pus  FROM  URETHRA, 
SHOWING  THE  Cocci 
WITHIN  A  LEUCOCYTE. 


1Neisscr,  Cent.  f.  d.  med.  Wiss.,  1879. 

2  Bumm,  ''Boitr.  z.  Kermtniss  des  Gonococcus, "  Wiesbaden,  1885. 

3  Sclioltz,  Arch.  f.  Dermat.,  1899. 

547 


548  PATHOGENIC   MICROORGANISMS 

and  others  to  take  place  in  the  free  secretions,  not  in  the  depth  of  the 
tissues.  The  intracellular  position,  which  is  of  considerable  diagnostic 
importance,  is  lost  to  a  great  extent  in  secretions  from  chronic  cases. 
In  smears  made  from  pure  cultures  the  arrangement  in  groups  of  two 
may  often  be  less  marked  than  in  pus,  clusters  of  eight  or  more  being 
common. 

The  gonococcus  is  non-motile  and  does  not  form  spores.  It  is  easily 
stained  with  aqueous  anilin  dyes.  Methylene-blue  alone,  or  eosin 
followed  by  methylene-blue,  give  good  results.  An  excellent  picture 
is  obtained  with  the  Pappenheim-Saathof  stain  consisting  of 

Methyl  green 0.15 

Pyronin     0.  5 

96  per  cent  alcohol 5.  0 

Glycerin    20.  0 

2  per  cent  carbolic  acid  water  ad 100.  0 

Fix;  stain  1-2  min. 

Gram's  method  of  staining,  however,  is  the  only  one  of  differential 
value,  gonococcus  being  Gram  negative.  The  Gram  stain  applied  to 
pus  from  the  male  urethra,  while  not  absolutely  reliable,  is,  for  practical 
purposes,  sufficiently  so  to  make  a  diagnosis.  In  exudates  from  the 
vagina  or  from  the  eye  the  morphological  picture  is  not  so  reliable, 

owing  to  the  frequent  presence  in 
these  regions  of  other  Gram-negative 
cocci.  The  great  scarcity  of  gono- 
cocci  in  very  chronic  discharges 
necessitates  thorough  cultural  investi- 
gation ;  negative  morphological  exami- 
nation in  such  cases  can  not  be 
regarded  as  conclusive.4 

Cultivation. — The  gonococcus  is 
delicate  and  difficult  to  cultivate. 
Bumm5  obtained  his  first  growths 

58.-GoNococcus.    Smear        uPon     human     blood     serum     which 
from  pure  culture.  had    been    heated    to   partial    coagu- 

lation. 

The  medium  most  commonly  used  at  the  present  day  was  intro- 
duced by  Wertheim,6  and  consists  of  a  mixture  of  two  or  three  parts  of 

4  Heiman,  Medical  Record,  1896. 

5  Bumm,  Dent.  med.  Woch.,   1885. 

6  Wertheim,  Arch.  f.  Gynakol.,  1892. 


t. 


•  *.• 


DIPLOCOCCUS  GONORRHOEA  549 

meat  infusion-agar  with  one  part  of  uncoagulated  human  ascitic  fluid, 
hydrocele  fluid,  or  blood  serum.  The  agar  is  melted  and  cooled  to  45° 
before  the  serum  is  added.  The  mixture  may  then  be  slanted  in  the 
test  tube  or  poured  into  a  Petri  plate.  One  per  cent  of  glucose  may  be 
added.  Cultures  in  fluid  media  may  be  obtained  by  similar  additions  of 
serum  to  meat-infusion-pep  ton-broth.  Whole  rabbit's  blood  added  to 
agar,  or  the  swine-serum-nutrose  medium  of  Wassermann  7  may  occa- 
sionally be  used  with  success. 

Plates  may  be  made  by  smearing  for  enrichment  a  drop  of  blood 
from  the  finger  over  the  surface  of  agar  in  the  manner  of  Pfeiffer's 
method  for  influenza-bacillus  cultivation.  Inoculations  from  gonorrheal 
material  are  best  made  by  surface  smearing  upon  plates,  since  the  gono- 
coccus  grows  best  in  the  presence  of  free  oxygen.  Growth  becomes  more 
luxuriant  after  prolonged  cultivation  upon  artificial  media.  The  most 
favorable  reaction  of  media  is  neutrality  or  slight  acidity. 

When  the  gonococcus  has  been  successfully  cultivated  from  pus  upon 
media  without  serum  additions,  the  success  has  probably  been  due  to 
the  substances  carried  over  in  the  pus.  The  ease  of  cultivation  differs 
considerably  with  different  strains  of  gonococci.  Some  grow  very 
heavily  after  first  isolation,  but  the  majority  show  a  very  delicate  growth 
even  on  rich  ascitic  glucose  agar.  After  several  generations  of  growth 
on  artificial  media,  however,  the  organism  develops  with  increasing  ease 
and  on  simpler  media.  It  may  eventually  be  cultivated  on  plain  agar, 
especially  when  this  is  made  of  veal  infusion.  Recently  a  medium  upon 
which  gonococci  after  first  cultivation  can  be  grown  with  ease  has  been 
recommended  by  Edward  B.  Vedder.8  The  medium  consists  of  a 
1.5  to  1.75  per  cent  agar  made  with  beef  infusion  neutral  to  phenol- 
phthalein,  and  after  clearing,  1  per  cent  of  corn  starch  added.  The  corn 
starch  is  best  added  after  grinding  with  a  little  agar  to  avoid  clumps, 
this  then  being  poured  into  the  bulk  of  the  agar  and  thoroughly  mixed. 
The  medium  should  be  sterilized  at  not  over  15  Ibs.  pressure  to  avoid 
changes  in  the  starch.  Recently  we  have  isolated  several  strains  of 
gonococci  which  grew  very  heavily  on  simple  media  without  ascitic 
fluid  in  the  second  culture  generation. 

The  gonococcus  will  develop  sparsely  under  anaerobic    conditions 

7  Wasserma-nn,  Berl.  klin.  Woch.,  1897. 

(Fifteen  c.c.  swine-serum,  35  c.e.  of  water,  3  c.c.  glycerin,  with  two  per  cent 
nutrose.  The  nutrose  is  dissolved  by  boiling  and  the  solution  sterilized.  This 
is  then  added  to  agar,  in  equal  parts,  and  used  in  plates.) 

8  Vedder,  Jour.  Infec.  Dis.,  May  15,  1915,  xvi,  385. 


550  PATHOGENIC   MICROORGANISMS 

but  has  marked  preference  for  aerobiosis.  The  optimum  temperature 
is  37.5°  C.  Growth  ceases  above  38.5°  and  below  30°. 

Upon  suitable  media  colonies  appear  as  extremely  delicate,  grayish, 
opalescent  spots,  at  the  end  of  twenty-four  hours.  The  separate  colo- 
nies do  not  tend  to  confluence  and  have  slightly  undulated  margins. 
Touched  with  a  platinum  loop  their  consistency  is  found  to  be  slimy. 
In  fluid  media,  growth  takes  place  chiefly  at  the  surface. 

Types  of  the  Gonococcus. — As  in  the  case  of  so  many  other  organisms, 
it  has  been  found  that  the  Gram-negative  diplococci  which  cause  gon- 
orrheal  infections  are  not  a  single  type,  but  must  be  regarded  as  rep- 
resenting a  group,  including  many  closely  related,'  but  antigenically 
differentiable  subgroups.  Such  subgrouping  of  species  formerly  regarded 
as  homogeneous  has  been  a  very  natural  development  of  the  more 
intensive  study  of  serum  reactions,  incident  to  diagnostic  agglutination 
and  complement  fixation,  and  to  the  control  of  specific  therapy.  Torrey9 
and  Teague  and  Torrey  1  °  in  1907  showed  that  the  gonococcus  group  is 
not  homologous,  but  that  agglutination  and  agglutinin  absorption 
divided  this  group  into  at  least  three  separate  subtypes.  Agglutinin 
absorption  seemed  to  show  more  types  than  complement  fixation,-  a 
matter  which  we  ourselves  would  rather  expect  at  the  present  day 
because  of  the  almost  universal  experience  that  complement  fixation 
reactions  are  not  as  strictly  specific  as  agglutination.  Torrey's  claims 
have  been  to  some  extent  misquoted  in  the  past  ten  years,  in  that  he 
himself  never  supposed  that  his  ten  strains  represented  the  entire  gon- 
ococcus group.  In  1910  Watabiki  n  made  a  similar  study  of  the  gon- 
ococcus group,  and  studying  a  limited  number  of  strains,  confirmed  the 
heterogeneous  nature  of  the  group  by  referring  to  them  as  "  compara- 
tive but  not  distinctive  differences  between  individual  strains."  In 
1915  Louise  Pearce 12  made  a  comparison  between  gonococci  isolated  from 
adult  males  and  from  the  vulvovaginitis  of  children.  She  came  to  the 
conclusion  that  strains  from  these  two  sources  constituted  fairly  definite 
serologically  distinct  groups,  that  at  least  there  was  a  relative  distinc- 
tion between  the  two  types.  This,  in  view  of  the  important  sanitary 
problem  involved  in  these  infections  in  children,  would  be  of  great 
importance  if  confirmed.  We  will  refer  to  it  again  below.  More 


9  Torrey,  Jour.  Med.  Res.,  16,  1907,  329. 

10  Teague  and  Torrey,  Jour.  Med.  Res.,  17,  1907,  223. 
»  Watabiki,  Jour.  Infec.  Dis.,  1910,  7,  159. 

12  Pearce,  Jour.  Med.  Res.,  21,  1915,  289. 


D1PLOCOCCUS  GONORRHCE^  551 

recently,  Hermanies 13  studied  85  gonococcus  strains  from  various 
sources,  using  for  cultivation  the  partial  tension  method  of  Wade  W. 
Oliver.  He  concluded  that  gonococci  fall  into  distinct  types,  with  little 
relationships  to  each  other.  Agglutinins  produced  by  one  type  cannot 
be  absorbed  by  strains  of  other  types,  and  his  85  strains  fell  into  six 
distinct  groups.  At  the  time  of  the  present  writing,  Torrey  is  again 
working  on  the  same  subject,  since  the  conflicting  evidence  of  these 
researches  and  earlier  work  shows  clearly  that  the  subject  cannot  be 
regarded  as  closed.  The  work  Torrey  is  doing  with  Buckell  is  not  yet 
completed,  and  it  would,  therefore,  be  rash  to  make  a  final  statement 
concerning  his  opinion,  but,  since  he  has  devoted  a  considerable  amount 
of  time  and  energy  to  this  subject,  and  since  his  bacteriological  judgment 
is  unusually  sound,  we  quote  as  follows  from  advanced  information 
he  has  courteously  furnished  us  in  a  personal  communication.  Tor- 
rey's  impressions  at  the  present  time  are  that  it  is  not  possible  to  demon- 
strate definite  groups,  such  as  the  three  fixed  pneumococcus  types,  or 
even  groupings,  comparable  to  that  established  for  the  meningococcus 
and  discussed  in  another  section.  A  study  of  50  strains  from  many 
sources  and  from  different  parts  of  the  world  by  agglutination  and 
agglutinin  absorption,  has  shown  that  there  are  certain  generalized 
strains  which  possess  antigenic  properties  common  to  a  considerable 
part  of  the  " whole"  species.  On  the  other  hand,  there  are  a  large 
number  of  variants  among  recently  isolated  strains  which  show  no 
serological  relationship  to  one  another  as  far  as  specific  agglutination  is 
concerned,  and  which  quite  often  show  some  specific  relationship  to 
one  or  more  of  the  specific  strains.  These  -  variants  overlap  and  show 
such  intergradations  that  it  is  not  possible  to  subdivide  them  into 
definite  groups.  It  has  also  been  noticeable  in  his  work  that  some  of  the 
original  strains  which  he  studied  as  long  as  fourteen  years  ago,  and  which 
at  that  time  seemed  to  be  unrelated  to  each  other,  are  now  beginning 
to  show  some  specific  relationships.  All  the  older  strains  show  greater 
relationships  than  do  similar  groups  of  recently  isolated  ones.  Torrey 
has  not  been  able  to  confirm  the  contentions  of  Louise  Pearpe  that  there 
is  a  recognizable  distinction  between  vulvovaginitis  strains  from  chil- 
dren and  those  from  adult  males. 

Recognition  of  the  Gonococcus. — Speaking  of  sugar  fermentations, 
Torrey  has  come  to  the  conclusion  that,  taken  together  with  typical 
colony  formation,  morphology,  staining  reactions  and  absence*  of  growth 
on  ordinary  laboratory  media,  sugar  fermentations  form  Ihe  most 


,  Jour.   In  foe.   Dis.,  28,   192],    1 :'.:'.. 


552  PATHOGENIC   MICROORGANISMS 

reliable  methods  of  recognition  of  the  gonococcus.  Of  60  strains  exam- 
ined by  him,  all  except  one  fermented  glucose,  and  none  of  them  fer- 
mented maltose,  thus  -differentiating  from  meningococcus.  The  one 
exception  was  an  old  strain,  all  the  recent  ones  fermenting  true  to  type. 

A  gonococcus  can,  therefore,  be  recognized  bacteriologically  by  the 
Gram-negative  diplococcus  form,  the  typical  colony  formation  on 
ascitic  agar,  its  failure  to  grow  on  simpler  media  or  at  room  temperature, 
its  ability  to  split  dextrose  and  failure  to  split  maltose. 

Resistance. — Recent  cultures  of  gonococcus,  if  not  transplanted, 
usually  die  out  within  five  of  six  days  at  incubator  temperature.  At 
room  temperature  they  die  more  rapidly. 

The  resistance  of  the  gonococcus  to  light  and  heat  is  slight.  A  tem- 
perature of  41°  to  42°  kills  it  after  a  brief  exposure.  Complete  drying 
destroys  it  in  a  short  time.  Incompletely  dried,  however,  and  pro- 
tected from  light  (gonorrheal  pus)  it  may  live,  on  sheets  and  clothing, 
for  as  long  as  eighteen  to  twenty-four  hours.14 

It  is  easily  killed  by  most  disinfectant  solutions  15  in  high  dilution 
and  seems  to  be  almost  specifically  sensitive  to  the  various  silver  salts, 
a  fact  of  therapeutic  importance. 

Pathogenicity. — Gonorrheal  infection  occurs  spontaneously  only 
in  man.  True  gonorrheal  urethritis  has  never  been  experimentally  pro- 
duced in  animals.  In  human  beings,  apart  from  the  infection  in  the 
male  and  female  genital  tracts,  and  in  the  conjunctive,  the  gonococcus 
may  produce  cystitis,  proctitis,  and  stomatitis.  It  may  enter  the  cir- 
culation, giving  rise  to  septicemia,16  to  endocarditis  and  arthritis.  Iso- 
lated cases  of  gonorrheal  periostitis  and  osteomyelitis  have  been 
reported.17 

The  acute  infections  of  the  genito-urinary  passages  are  often  fol- 
lowed by  prolonged  chronic  infection,  which,  though  quiescent,  may  for 
many  years  be  a  source  of  social  danger.  In  children,  especially  females, 
the  infection  is  not  rare,  and  may  assume  epidemic  characters,  traveling 
from  bed  to  bed  in  institutions.  Such  hospital  epidemics  can  be  stopped 
only  by  the.  most  rigid  isolation.  This  is  more  specifically  dealt  with 
in  the  paragraphs  on  Sanitary  Considerations,  below. 

While  inoculation  of  animals  has  never  resulted  in  active  prolifera- 
tion of  the  gonococcus  upon  the  new  host,  local  necrosis,  suppuration, 


14  Heiman,  Medical  Eecord,  1896. 

15  Schaeffcr  und  Steinschn eider,  Kong.  Deut.  Dermat.  Geselis.,  Breslau,  1894. 
16Eeview  of  cases  of  Gon.  Septicemia,  Faure-Beaulieu,  Thesis,  Paris,   1906. 
>7  Ullmann,  Wien.  med.  Presse,  1900. 


DIPLOCOCCUS  GONORRHCEjE  553 

and  temporary  systemic  reactions  have  been  produced  by  subcutaneous 
and  intraperitoneal  inoculation.  A  toxin  has  been  isolated  by  Niko- 
laysen  18  by  extraction  from  the  bacterial  bodies  with  distilled  water  or 
sodium  hydrate  solutions.  It  was  found  to  be  resistant  to  a  tempera- 
ture of  120°  and  to  remain  potent  after  complete  drying.  The  same 
author  found  that  the  isolated  toxin  and  dead  cultures  were  fully  as 
toxic  for  animals  as  living  cultures,  0.01  gram  killing  a  white  mouse. 

Specific  injury  to  the  nervous  system  by  injections  of  gonococcus 
toxin  has  been  reported  by  Moltschanoff.19 

The  secretion  of  a  true  soluble  toxin  by  the  gonococcus,  asserted  by 
Christmas,20  is  denied  by  Wassermann,21  Nikolaysen,22  and  others. 
Christmas,23  and,  more  recently,  Torrey,24  have  reported  successful 
active  immunization  of  animals  by  repeated  injections  of  whole 
bacteria.  Torrey  and  others  apparently  have  successfully  treated 
human  cases  by  injections  of  the  serum  of  immunized  animals. 

Antibodies  to  Gonoooccus. — Patients  infected  with  gonococci 
seem  to  produce  antibodies  against  the  organisms.  Although  in  the 
ordinary  gonorrheal  urethritis,  or  vaginitis,  it  is  relatively  simple  to  make 
the  diagnosis  by  finding  gonococci  in  the  discharges,  diagnosis  may  be 
difficult  in  cases  of  gonorrheal  rheumatism,  or  endocarditis,  when 
isolation  of  the  bacteria  fails  or  when  the  connection  between  the  local 
venereal  disease  and  the  general  condition  is  obscure.  Various  sero- 
logical  diagnostic  methods  have  been  attempted,  and  of  recent  years 
the  complement-fixation  test  has'  been  found  to  be  very  useful.  The 
method  has  been  especially  developed  by  Archibald  McNeil,  at  the 
New  York  Department  of  Health.  It  consists  in  making  a  polyvalent 
antigen,  using  the  10  Torrey  strains  which  are  kept  in  stock  transplants 
on  glucose  ascitic  agar.  It  has  been  found  that  the  best  medium  for 
antigen  production  is  an  agar  made  of  "bob  veal."  For  the  production 
of  antigen,  stock  cultures  are  transplanted  on  "bob  veal"  agar,  without 
salt,  glucose  or  ascitic  fluid,  the  reaction  carefully  adjusted  to  an  acidity 
of  0.1  per  cent  to  0.2  per  cent.  Twenty-four  hour  growths  on  this 
medium  are  scraped  off  and  emulsified  in  neutral  sterile  distilled  water. 
The  emulsion  is  autolyzed  one  hour  in  a  water  bath  at  56°  and  heated 

™NiTcolaysen,  Cent.  f.  Bakt.,  1897. 
"Moltschanoff,  Munch,  med.  Woch.,  1899. 

20  Christmas,  Ann.  de  1  'Inst.  Pasteur,  1897. 

21  Wassermann,  Zeit.  f.  Hyg.,  xxvii,  1897. 

22  Nikolaysen,  Fort.  d.  Med.,.xxi,  1897. 

23  Christmas,  loc  cit. 

24  '±orrey^  Jour.  Amer.  Med.  Assn.,  xlvi,  1906. 


554  PATHOGENIC   MIRCOORGANISMS 

one  hour  at  80°  C.  It  is  then  filtered  through  a  sterile  Bcrkefcld  filter. 
The  filtrate  is  aseptically  bottled  and  sterilized  three  days  at  56°,  half  an 
hour  each  day.  It  is  then  made  isotonic  and  is  ready  for  titration. 

Vaccine  therapy  in  systemic  gonorrheal  infection  has  been  tried 
and  is  promising/  though  as  yet  unconvincing.  The  vaccine,  if  possible, 
should  be  made  with  the  organism  isolated  from  the  patient,  for  reasons 
described  above.  Passive  immunization  with  the  serum  of  gonococcus- 
immune  animals  has  also  been  attempted,  but  records  on  it  at  present 
are  not  sufficiently  complete  to  permit  definite  judgment. 

SANITARY  CONSIDERATIONS  IN  CONNECTION  WITH 
GONOCOCCUS  INFECTION 

Of  the  three  prevalent  venereal  infections,  those  caused  by  the 
gonococcus  are  probably  the  most  common.  For  more  exhaustive 
statistical  studies  of  the  prevalence  of  these  diseases  the  reader  is  referred 
to  such  books  as  those  of  Pusey,  Morrow,  and  the  larger  text-books  of 
hygiene,  such  as  that  of  Rosenau.  Although  it  has  been  well  known 
that  gonorrhea  was  extremely  common,  the  astonishing  prevalence 
among  young  men  of  draft  age  was  revealed  during  the  late  war  when 
the  figures  of  the  Surgeon  General25  show  that  about  5.6  per  cent  of 
the  men  who  came  into  the  military  service  were  infected  with  a  veneral 
disease.  Considering  that  these  diseases,  in  their  early  detectable  acute 
stages,  do  not  last  very  long,  that  many  cases  still  apparent  to  a  slight 
degree  must  surely  be  missed  in  physical  examinations  of  large  numbers 
of  men,  it  seems  to  indicate  that  the  estimate  by  many  authorities  of 
a  prevalence  of  venereal  disease  in  civilian  life  as  high  as  10  per  cent 
may  be  very  near  the  truth.  The  percentage  of  gonorrhea  to  other 
venereal  diseases  is  probably  pretty  well  exemplified  by  the  percentage 
of  these  diseases  for  the  entire  army  during  1918,  during  which,  accord- 
ing to  the  Surgeon  General's  report,  there  were  44,213  cases  of  syphilis, 
16,173  cases  of  chancroid,  and  167,475  cases  of  gonococcus  infection,  a 
total  of  227,861  cases  of  venereal  disease.  The  rate  for  syphilis  was 
17.56  per  1000,  for  chancroid  6.42,  for  gonorrhea  66.50. 

One  of  the  great  dangers  in  connection  with  gonorrheal  infection  has 
been  the  relative  indifference  of  the  public  to  these  diseases.  In  the 
past,  there  has  been  a  remarkable  lack  of  appreciation  of  the  seriousness 
of  the  infection,  which  actually,  in  its  economic  and  sociological  impor- 

23  Rep.  of  Surgeon  General,  U.  S.  A.,  1919,  Vol.  1,  p.  956. 


GONOOOCCUS  INFECTION  555 

tance,  is  equal  to,  if  not  more  serious,  than  syphilis.  The  gonococcus  is 
primarily  infectious  for  the  genital  organs,  but  may  also  infect  the 
eye,  and  in  its  secondary  manifestations  cause  disease  of  the  prostate, 
epidydimis  and  bladder  of  the  male,  of  the  fallopian  tubes  and  ovaries 
of  the  female.  In  both  it  may  and  often  does  cause  sterility.  Invad- 
ing the  blood  stream,  it  may  cause  endocarditis,  and  not  infrequently 
an  acute  and  .subacute  arthritis  which  is  characterized  by  its  frequent 
localization  in  single  joints  or  the  bursse  about  joints,  and  cause  peri- 
articular  inflammation.  It  may,  but  rarely  does  attack  other  organs. 

A  most  important  consideration  is  the  difficulty  of  complete  cure. 
A  male  who  has  contracted  gonorrhea  may  seem  to  be  completely 
cured,  but  if  a  posterior  urethritis  has  occurred,  the  organisms  may 
•remain  viable  and  capable  of  infecting  others  for  a  great  many  years. 
Individuals  who,  therefore,  seem  to  have  been  cured  for  years,  may 
still  cause  infection  upon  marriage,  a  fact  which  is  the  most  frequent 
cause  of  gynecological  lesions  in  women.  It  goes  without  saying  that 
even  the  most  careful  bacteriological  examination  of  such  individuals 
may  often  fail  to  reveal  the  gonococci,  even  though  they  may  be  present. 

Infection  with  gonococcus  is  almost  invariably  by  sexual  contact, 
though  the  organism  may  remain  viable  on  wearing  apparel,  bed  cloth- 
ing, towels,  hands,  etc.,  for  brief  periods,  especially  if  protected  from 
light  and  drying,  and  others  may  be  infected  in  this  way.  The  danger 
of  self-infection  of  eyes  by  people  who  are  suffering  from  an  acute  dis- 
charge is  a  frequent  one,  and  physicians  and  nurses,  especially,  are  liable 
to  such  infection. 

Gonorrheal  infection  of  the  eye  is  one  of  the  most  serious  infections 
that  can  occur  in  this  organ.  Ophthalmia  of  the  new  born  may  be  due 
to  other  organisms,  but  is  almost  invariably  caused  by  the  gonococcus. 
It  is  acquired  by  the  child  in  the  course  of  delivery,  from  the  secretions 
of  the  mother,  and  if  not  attended  to,  may  lead  to  blindness.  The 
importance  of  this  infection  may  be  estimated  from  the  following 
figures  quoted  by  Rosenau  from  Kerr,  who  states  that  in  the  United 
States  and  Canada  23.9  per  cent  of  351  admissions  to  schools  for  the 
blind  in  1910  the  blindness  was  the  result  of  gonorrheal  infection. 

Fortunately,  the  method  introduced  by  Crede  has,  to  a  very  large 
extent,  done  away  with  this  accident.  Crede,  many  years  ago,  intro- 
duced the  method  of  instilling  a  2  per  cent  silver  nitrate  solution  into 
the  conjunctiva]  sa.cs  of  every  child  at  birth.  Since  his  time  oilier  silver 
salts  have  been  in  use,  the  most  popular  ones  at  the  present  time  being 
protargol,  5  per  cent  solution,  and  argyrol,  20  per  cent  solution,  which 


556  PATHOGENIC   MICROORGANISMS 

are  dropped  into  the  eye  at  birth.  It  is  extremely  important  that  this 
should  be  done  properly  and  the  entire  conjunctival  sac  bathed  in  the 
fluid.  The  method  is  so  important  that  it  is  regarded  as  a  matter  of 
very  serious  and  inexcusable  omission,  if,  under  any  circumstances,  in 
dealing  with  any  class  of  the  population,  the  physician  managing  a 
childbirth  fails  to  carry  out  this  measure  as  soon  as  feasible  after 
birth. 

Another  very  important  gonorrheal  problem  is  the  vulvovangitis 
which  occurs  in  children.  In  our  own  experience,  this  infection  has  oc- 
curred most  often  in  connection  with  the  children's  wards  in  hospitals. 
The  condition  has,  however,  been  observed  in  schools  and  in  small  family 
groups  where  children  were  infected  by  sleeping  in  the  same  beds  with 
adults.  In  hospitals  the  disease  may  spread  in  epidemics,  and  from 
bed  to  bed,  with  an  ease  that  is  astonishing  when  one  considers  the 
delicate  life  of  the  gonococcus  outside  the  body.  It  has  often  been 
extre  nely  difficult  to  stop  such  bed  to  bed  infection,  in  spite  of  the  most 
rigid  precautions.  Epidemics  are  so  difficult  to  arrest,  and  the  conse- 
quences for  the  child  so  grave  from  many  points  of  view,  that  it  has 
becone  the  custom  in  all  well-managed  hospitals  to  delay  the  admission 
of  female  children  to  the  general  children's  ward  until  vaginal  smears 
have  been  made  and  examined  for  gonococci.  It  is  in  our  opinion 
extremely  important  that  when  such  smears  are  made,  they  should  be 
taken  not  only  from  the  visible  secretion,  but  should  be  taken  from  high 
up  in  the  vagina  through  a  small  Kelly  speculum,  with  good  illumina- 
tion. When  there  is  danger  of  spread  and  a  case  has  been  inadver- 
tently admitted,  only  the  greatest  care  in  avoiding  indirect  contact  from 
bed  to  bed  can  stop  it.  As  a  matter  of  routine  in  children's  wards, 
there  should  be  separate  thermometers,  unless  all  thermometers  are  very 
carefully  sterilized,  thermometers  should  be  kept  in  weak  carbolic 
solutions  and  washed  with  alcohol  before  use;  the  sterilization  of  diapers 
and  towels  should  be  attended  to,  nurses  handling  cases  with  discharge 
should  wear  gloves,  and  there  should  be  no  common  use  of  towels  and 
washing  utensils.  Great  attention  should  be  given  to  the  scouring  of 
bath  tubs,  and  bed  linen,  night  clothes,  etc.,  should  be  sterilized  by 
boiling. 

Public  Health  Management  of  Venereal  Diseases. — During  the 
past  ten  years  there  has  been  a  very  wholesome  increase  of  interest  in 
venereal  disease  prevention.  There  are  certain  general  fundamental 
principles  which  apply  to  all  venereal  diseases  equally.  In  the  first 
place,  it  is  necessary  to  look  upon  venereal  infections  as  preventable 


GONOCOCCUS   INFECTION  557 

diseases.  It  has  been  unfortunate  that  the  sanitary  and  moral  issues 
have  been  so  closely  interwoven  in  these  diseases,  that  it  has  been 
impossible  to  create  the  free  discussion  and  spread  the  information 
necessary  to  obtain  the  cooperation  of  the  public  in  these  matters. 
Without  public  education  and  cooperation,  large  scale  public  health 
results  cannot  be  achieved.  In  our  opinion,  one  of  the  most  important 
factors  that  have  prevented  earlier  progress  in  the  prevention  of  venereal 
disease  has  been  the  ignorance  of  the  public  in  regard  to  these  matters. 
It  has  been  especially  wrong  that  women  of  the  marriageable  age  have 
been  kept  in  ignorance  about  facts  concerning,  these  infections,  an 
ignorance  which  has  often  left  them  absolutely  at  the  mercy  of  chance. 
Accurate  and  clear  information,  free  of  sensationalism,  will  do  more 
eventually  to  reduce  the  venereal  rate  than  any  other  single  factor. 

It  is  not  the  function  of  a  book  of  this  kind  to  go  into  the  very  com- 
plex problems  of  general  sex  education,  and  the  moral  issues  involved. 
We  will  restrict  ourselves  entirely  to  the  purely  sanitary  phases  of  the 
problem.  Chief  among  these  are : 

1.  Diagnosis. — Education  and  knowledge  of  the  seriousness  of  these 
infections  should  lead  to  a  gradual  attraction  of  patients,  away  from 
quacks,  to  reliable  clinics  and  physicians.     The  development  of  diag- 
nostic clinics  by  departments  of  health,  the  improvement  of  clinical 
facilities  in  large  cities,  and  the  better  understanding  by  physicians,  as 
a  whole,  of  the  sanitary  importance  of  these  relatively  simple  infections, 
must  lead  to  more  accurate  diagnosis  and  proper  instruction  of  the 
patient. 

2.  Reporting  of  Venereal  Diseases. — In  a  great  many  communities 
at  the  present  time  gonorrheal  infections,  as  well  as  other  venereal 
diseases,  are  regarded,  like  other  communicable  diseases,  as  subject  to 
report.     There  are  many  reasons  why  such  reporting  systems  will  meet 
with  objections,  and  will   for   many  years   be   unsatisfactory.     This, 
too,  we  believe  is  a  matter  of  education,  and  the  fact  that  it  will  fail  for 
the  present  is  no  reason  why  the  principle  should  not  be  upheld.     Event- 
ually we  believe  it  will  be  accepted  as  a  sensible  and  necessary  step. 
These  diseases  are  communicable  to  others  during  certain  stages,  and 
when  they  are  regarded  primarily  as  possibilities  for  the  spread  of  dis- 
ease and  the  public  stress  is  not  laid  purely  on  the  moral  issue,  objections 
to  reporting  will  cease.     In  our  opinion  the  chief  objection  that  has 
been  raised  against  the  reporting  of  these  diseases  is  the  permanent 
record,    apparent    disgrace    and    perhaps    opportunity    for   blackmail 
which  is  opened  by  the  public  registration  of  an  individual  in  this  way. 


558  PATHOGENIC   MICROORGANISMS 

When  we  consider,  on  the  other  hand,  these  dangers  as  balanced  against 
the  danger  of  the  uncontrolled  circulation  among  their  fellows  of  indi- 
viduals capable  of  infecting  others,  there  seems  very  little  choice  in  our 
minds  between  the  two  evils.  Moreover,  we  believe  it  would  be  possible 
to  develop  a  system  of  reporting  whereby  the  reported  individual  could 
have  the  record  destroyed  when  he  could  bring  a  certificate  of  cure  from 
a  responsible  clinic  or  physician.  This,  we  believe,  would  add  a  further 
inducement  to  proper  care  and  cure.  At  any  rate,  we  believe  that  the 
prompt  report  of  cases,  following  them  up  from  municipal  health 
bureaus,  and  prompt  destruction  of  the  record  when  the  individual  has 
been  cured,  will  greatly  aid  in  this  matter. 

3.  H capitalization. — It  will  probably  be  impossible  to  hospitalize 
all  infectious  cases  of  venereal  diseases  because,  unfortunately  from 
the  public-health  point  of  view,  these  patients  are  not  incapacitated 
during  their  most  infectious  stages.  We  are  able  to  confine  a  case  of 
smallpox  with  or  without  consent,  but  diseases  that  in  their  remote 
possibilities  are  responsible  for  far  greater  injury  and  unhappiness,  are 
permitted  to  walk  about  and  follow  their  own  devices,  through  the 
course  of  their  illnesses.  The  eventual  ideal  would  consist  in  making 
physicians  responsible  for  the  isolation  of  cases  which  came  under 
their  care  and  to  hospitalize  those  who  could  not  be  taken  care  of  in 
their  own  quarters.  Hospitalization  in  separate  hospitals  would  confer 
so  great  a  stigma  that  it  would  probably  be  impossible.  It  might  also 
be  impossible  to  admit  these  cases  into  general  hospitals  in  spite  of 
special  arrangements.  We  do  not  ourselves  believe  that  compulsory 
hospitalization  could  be  enforced  at  the  present  time.  It  should  be 
looked  upon,  however,  as  an  attempt  worth  making,  as  soon  as  educa- 
tion and  general  public  cooperation  has  reached  a  point  at  which  success 
would  seem  at  least  not  totally  out  of  question.  It  is  our  opinion  that 
the  sooner  the  attitude  toward  these  diseases  is  made  one  purely  of 
sanitary  principles,  and  the  more  purely  moral  factors  are  allowed 
to  take  care  of  themselves  under  the  influence  of  increased  civilization 
and  sense  of  community  responsibility,  the  sooner  these  ends  may  be 
accomplished. 

While  it  is  of  course  quite  impossible  to  do  justice  to  as  funda- 
mentally important  a  problem  as  the  sanitary  control  of  venereal  dis- 
eases in  a  section  of  this  kind,  it  has  seemed  to  us  of  great  importance 
to  at  least  point  out  to  physicians  and  bacteriologists  who  may  read 
this  book,  the  enormous  responsibility  that  falls  upon  them  whenever 
they  are  in  a  position  to  deal  with  cases  of  this  kind. 


(joNororrrs  IN  FICTION  559 

Prophylaxis  of  Gonorrhea. — As  practiced  in  the  United  States 
army  stations  during  the  war26  this  consisted  in  injecting  about  10  c.c. 
of  a  2  per  cent  protargol  into  the  urethra — enough  to  thoroughly  dis- 
tend it,  with  a  glass  hand  syringe,  holding  it  there  with  the  syringe  in 
place,  for  one-half  minute.  The  procedure  is  twice  repeated.  Its 
success  depends  very  largely  upon  early  application  after  intercourse. 
As  to  general  efficiency  in  regard  to  gonorrhea  we  are  not  in  a  position 
as  vet  to  submit  reliable  statistics. 


MICROCOCCUS    CATARRHALIS 

Micrococcus  catarrhalis  is  a  diplococcus  described  first  by  R. 
Pfeiffer,27  who  found  it  in  the  sputum  of  patients  suffering  from  catarrhal 
inflammations  of  the  upper  respiratory  tract.  It  was  subsequently 
carefully  studied  by  Ghon  and  H.  Pfeiffer.28  According  to  these  authors 
the  pathogenic  significance  of  the  micrococcus  is  slight,  though  occasion- 
ally it  may  be  regarded  as  the  causative  factor  in  catarrhal  inflammations. 
Its  chief  claim  to  attention,  however,  lies  in  its  similarity  to  the  meningo- 
coccus  and  the  gonococcus,  from  neither  of  which  it  can  be  morpholog- 
ically distinguished.  It  is  decolorized  by  Gram's  stain,  appears  often  in 
the  diplococcus  form,  and  has  a  tendency,  in  exudates,  to  be  located 
intracellularly.  Not  unlike  the  two  microorganisms  mentioned,  too, 
it  shows  but  slight  pathogenicity  for  animals. 

Differentiation  from  gonococcus  is  extremely  simple  in  that  Micro- 
coccus  catarrhalis  grows  easily  on  simple  culture  media  and  shows 
none  of  the  fastidious  cultural  requirements  of  the  gonococcus. 

From  meningococcus  the  differentiation  is  less  simple  and,  because 
of  the  presence  of  both  microorganisms  in  the  nose,  is  of  great  impor- 
tance. 

Distinction  between  the  two  is  made  entirely  upon  cultural  charac- 
teristics and  agglutination  reactions.  Culturally,  Micrococcus  catar- 
rhalis grows  more  heavily  than  meningococcus  upon  the  ordinary 
culture  media.  The  colonies  of  Micrococcus  catarrhalis  are  coarsely 
granular  and  distinctly  white  in  contradistinction  to  the  finely  granu- 
lar, grayish  meningococcus  colonies.29  Micrococcus  catarrhalis  will 

26  Bayard  Clark,  Medical  Times,  April,   191!). 
-' Vliifif/<;   "J)ie   Mikroorg.,"    'M  <'<!.,   1896. 
2S  Ghon  uml  //.  Pfeiffer,  Zeit.  f.  klin.  Mcd.,  1902. 
-'•'  Ghon   mid   Pfeiffer,  loc.  cit. 


560  PATHOGENIC   MICROORGANISMS 

develop  at  temperatures  below  20°  C.,  while  meningococcus  will  not 
grow  at  temperatures  below  25°  C.30 

Dunham,31  who  has  recently  made  a  comparative  study  of  meningo- 
coccus and  other  Gram-negative  diplococci  from  the  nose  and  throat, 
states  that  while  some  of  the  supposed  Micrococcus  catarrhalis  cultures 
are  easily  distinguished  from  meningococcus  simply  by  the  character- 
istics of  their  growths  upon  two-per-cent  glucose  agar,  others  offer  great 
difficulties  to  differentiation.  He  recommends  as  a  differential  medium 
a  mixture  of  sheep  serum  and  bouillon  containing  1  per  cent  of  glucose. 
Upon  this  medium  all  true  meningococci  produce  acid,  but  no  coagula- 
tion, within  twenty-four  hours.  Cultures  from  the  nose  and  throat, 
however,  produce  acid  and  coagulation,  or  else  produce  an  alkaline 
reaction. 

30  Weichselbaum,  in  Kolle  und  Wassermann,  Bd.  iii,  p.  269. 

31  Dunham,  Jour.  Inf.  Dis.,  1907. 


CHAPTER  XXVIII 

BACILLUS     D1PHTHEKI.E,     BACILLUS    HOFFMANNI,    AND    BACILLUS 

XEBOSIS 

BACILLUS    DIPHTHERLffi 

SINCE  1821,  when  Bretonneau  of  Tours  published  his  observations, 
diphtheria  has  been  an  accurately  recognized  clinical  entity.  Our 
knowledge  of  the  disease  in  the  sense  of  modern  bacteriology,  however, 
begins  with  the  first  description  of  Bacillus  diphtheria  by  Klebs  in  1883. 
Klebs  1  had  observed  in  the  pseudomembranes  from  diphtheritic  throats, 
bacilli  which  in  the  light  of  more  recent  knowledge  we  can  hardly  fail 
to  recognize  as  the  true  diphtheria  organism.  His  work,  however,  was 
purely  morphological  and,  therefore,  inconclusive.  One  year  after 
this  announcement,  Loeffler  2  isolated  and  cultivated  an  organism  which 
corresponded  in  its  morphological  characters  to  the  one  described  by 
Klebs.  He  obtained  it  from  thirteen  clinically  unquestioned  cases 
of  diphtheria,  and,  by  inoculating  it  upon  the  injured  mucous  surfaces 
of  animals,  succeeded  in  producing  lesions  which  resembled  closely  the 
false  membranes  of  the  human  disease.  His  failure  to  find  the  bacillus 
in  all  the"  cases  he  examined,  his  finding  it,  in  one  instance,  in  a  normal 
throat,  and  his  inability  to  explain  to  his  own  satisfaction  some  of  the 
systemic  manifestations  of  the  infection  which  we  now  know  to  be  due 
to  the  toxin,  caused  him  to  frame  his  conclusions  in  a  tone  of  the  utmost 
conservatism.  The  second  and  third  publications  of  Loeffler,3  however, 
and  the  inquiry  into  the  nature  of  the  toxins  produced  by  the  bacillus, 
published  in  1888  by  Roux  and  Yersin,4  eliminated  all  remaining  doubt 
as  to  the  etiological  relationship  existing  between  this  organism  and  the 
disease. 

Innumerable  observations,  both  clinical  and  bacteriological,  by  other 
workers,  have,  since  that  time,  confirmed  the  early  investigations, 
and  it  is  to-day  a  scientific  necessity  to  find  the  bacillus  of  Klebs  and 

1  Klebs,  Verh.  d.  2.  Kongr.  f.  inn.  Medizin,  Wiesbaden,  1883. 

-Loeffler,  Mittheil.  a.  d.  kais.  Gesundheitsamt,  1884. 

3  Loeffler,  Cent.  f.  Bakt.,  1887  and  1890. 

*Roux  and  Yersin,  Ann.  de  1'inst.  Pasteur,  1888  and  1889. 

561 


562 


PATHOGENIC   MICROORGANISMS 


Loeffler  in  the  lesion  before  a  diagnosis  of  "diphtheria"  can  properly 
be  made. 

Morphology  and  Staining. — While  Bacillus  diphtherias  presents 
certain  characteristic  appearances  which  facilitate  its  recognition,  it  is, 
at  the  same  time,  subject  to  a  number  of  morphological  variations  with 
all  of  which  it  is  important  to  be  familiar.  These  variations  are,  to  a 
limited  extent,  dependent  upon  the  age  of  the  culture  and  upon  the 
constitution  of  the  medium  on  which  it  has  been  grown.  These  factors, 
however,  do  not  control  the  appearance  of  the  organism  with  any  degree 
of  regularity,  and  any  or  all  of  its  various  forms  may  occur  in  one  and 


*      *"Cr\*3&N*V          •»"  *v,v-.,  ^     <"^:    r   -  ^    ^-A^.,;-    v 

FIG.  59. — BACILLUS  DIPHTHERIA. 


the  same  culture.  It  is  likely  that  these  different  appearances  represent 
stages  in  the  growth  and  degeneration  of  the  individual  bacilli,  but  there 
does  not  seem  to  be  any  just  reason  for  believing  that,  as  several  observ- 
ers have  stated,  there  is  definite  correlation  between  its  microscopic 
form  and  its  biological  characteristics,  such  as  virulence,  toxicity,  etc. 

The  bacilli  are  slender,  straight,  or  slightly  curved  rods.  In  length 
they  vary  from  1.2  micra  to  6.4  micra,  in  breadth  from  0.3  to  1.1.  As 
seen  most  frequently  when  taken  from  the  throat  they  are  about  4  to 
5  micra  in  length.  They  are  rarely  of  uniform  thickness  throughout 
their  length,  showing  club-shaped  thickening  at  one  or  both  ends. 
Occasionally  they  may  be  thickest  at  the  center  and  taper  toward  the 
extremities.  When  thickened  at  one  end  only,  a  slender  wedge-shape 


BACILLUS   DIPHTHERIA  563 

results.  Such  forms  are  usually  straight,  of  smaller  size  than  their 
neighbors,  and  are  more  often  stained  with  great  uniformity.  These 
are  spoken  of  by  Beck5  as  the  "ground  type,"  and  assumed,  for  insuf- 
ficient reasons,  to  be  the  young  individuals.  Branched  forms  have  been 
described  by  some  investigators.  They  are  rare  and  probably  to  be 
regarded  as  abnormal  or  involution  forms  due  to  unfavorable  environ- 
ment. 

The  organisms  stain  with  the  aqueous  anilin  dyes.  A  characteristic 
irregularity  of  staining  which  is  of  great  aid  in  diagnosis  is  best  obtained 
with  Loeffler's  "alkalin  methylene-blue."  (For  preparation  see  section 
on  Staining,  p.  115.)  Stained  with  this  solution  for  five  to  ten  minutes 
many  of  the  bacilli  appear  traversed  by  unstained  transverse  bands 
which  give  them  a  striped  or  beaded  appearance.  The  longer  indi- 
viduals often  have  a  strong  resemblance  to  short  chains  of  strepto- 
cocci. Others  may  appear  unevenly  granular.  In  cultures  which  are 
about  eighteen  hours  old,  many  of  the  bacilli  may  show  deeply  stained 
oval  bodies  situated  most  frequently  at  the  ends.  These  are  the  so- 
called  "polar"  or  " Babes-Ernst "  bodies.6  Special  stains  have  been 
devised  for  the  demonstration  of  these  appearances.  One  of  these  was 
originated  by  Neisser,7  who  claims  for  it  differential  value  in  distinguish- 
ing these  organisms  from  pseudodiphtheria  and  xerosis  bacilli. 

His  method  requires  two  solutions : 

1.  Methylene  blue    (Griibler)    1  gram. 

Alcohol,  96  per  cent 20      c.c. 

Glacial  acetic  acid 50       ' ' 

Water    950       ' ' 

2.  Bismarck  brown    2  grams. 

Water    1,000      c.c. 


The  cover-slip  preparation,  after  having  been  fixed,  is  stained  with 
solution  No.  1  for  one  to  three  seconds.  It  is  then  washed  in  water  and 
immersed  for  from  three  to  five  seconds  in  solution  No.  2.  With  this 
stain  the  bodies  of  the  bacilli  appear  brown,  the  polar  granules  blue. 

Our  own  choice  of  a  stain  is  toluidin  blue.     A  staining  solution  can 
be  made  up  as  follows  according  to  the  original  formula,  we  believe,  of 

5  Beck,  in  Kollc  und  Wassermann,  ii,  p.  773. 

6  Babes,  Zeit.  f.  Hyg.,  Bd.  v,  1889. 

7  Neisser,  Zeit,  f.  Hyg.,  xxiv,  1897. 


564  PATHOGENIC   MICROORGANISMS 

Trincas,  and  quoted  from  the  First  Volume  of  the  Kolle  and  Wasser- 
man  Handbook. 

Toluidin  blue   0.25  gram 

Alcohol    5          c.e. 

Two  per  cent  acetic  acid 100          c.c. 

Staining  with  this  for  one  minute  gives  very  much  the  same  picture 
as  Loeffler's  alkalin-methylene-blue,  but  rather  a  clearer  contrast 
between  polar  bodies  and  cytoplasm,  and  makes  an  eminently  satis- 
factory stain. 

The  significance  of  the  polar  bodies  is  not  well  understood.  Their 
discoverer,  Ernst,  regarded  them  as  bodies  analogous  to  the  spores  of 
other  organisms.  The  ease  with  which  they  are  stained,  however,  and 
the  low  temperatures  to  which  the  bacteria  succumb  make  this  appear 
very  unlikely.  A  more  probable  interpretation  seems  to  be  that  of 
Escherich  8  who  regards  them  as  chromatic  granules. 

Stained  by  Gram's  method,  the  diphtheria  bacilli  retain  the  gentian- 
violet. 

In  stained  smears  from  the  throat  or  from  cultures  a  characteristic 
grouping  of  the  bacilli  has  been  observed.  They  lie  usually  in  small 
clusters,  four  or  five  together,  parallel  to  each  other,  or  at  sharp  angles. 
Two  organisms  may  often  be  seen  attached  to  each  other  by  their  cor- 
responding ends  while  their  bodies  diverge  to  form  a  "  V"  or  "  Y"  shape. 

Biological  Characteristics. — The  diphtheria  bacillus  is  a  non- 
motile,  non-flagellated,  non-spore-forming  ae'robe.  Its  preference  for 
oxygen  is  marked,  but  it  will  grow  in  anaerobic  environment  in  the  pres- 
ence of  suitable  carbohydrates.  It  does  not  liquefy  gelatin.  The 
bacillus  grows  at  temperatures  varying  between  19°  C.  and  42°  C.,  the 
most  favorable  temperature  for  its  development  being  37.5°  C.  Tem- 
peratures above  37.5°,  while  not  entirely  stopping  its  growth,  impede 
the  development  of  its  toxin. 

Resistance. — The  thermal  death  point  of  this  organism  is  58°  C. 
for  ten  minutes,  according  to  Welch  and  Abbott.  Boiling  kills  it  in 
about  one  minute.  Low  temperatures,  and  even  freezing,  are  well 
borne.  Desiccation  and  exposure  to  light  are  not  so  fatal  to  this  organ- 
ism as  to  most  of  the  other  pathogenic  bacteria.  Sternberg9  has  found 
it  alive  in  dried  bits  of  the  pseudomembrane  after  fourteen  weeks.  It  is 

8  Escherich,  ' '  Aetologie,  etc.,  d.  Diphth., ' '  Wien,  1894. 
0  Sternberg,  "  Manual  Bac.,  p.  455- 


BACILLUS  DIPHTHERIA  565 

easily  killed  by  chemical  disinfectants  in  the  strengths  customarily 
employed.  H2O2  seems  especially  efficacious  in  killing  the  organisms 
rapidly. 

Cultivation. — The  diphtheria  bacillus  grows  readily  on  most  of 

the  richer  laboratory  media.  It  will  grow  upon  media  made  of  meat 
extract,  but  develops  more  luxuriantly  on  all  those  which  have  a  meat 
infusion  as  their  basis.  While  it  will  grow  upon  both  acid  and  alkalin 
media,  it  is  sensitive  to  the  extremes  of  both,  the  most  favorable  reaction 
for  its  development  being  probably  about  0.5  per  cent  alkalinity  ex- 
pressed in  terms  of  N/l  NaOH.  Animal  proteins  added  to  the  media,  in 
the  form  of  blood  serum,  ascitic  fluid,  or  even  whole  blood,  increase 
greatly  the  rapidity  and  richness  of  its  growth.  Horse  serum  is  supposed 
by  some  to  be  especially  favorable.10 

Loeffler's  Medium. — The  most  widely  used  medium  for  the  cultiva- 
tion of  this  bacillus  is  the  one  devised  by  Loeffler.  This  consists  of; 

Beef  blood  serum 3  parts 

One  per  cent  glucose  meat-infusion  bouillon 1  part 

The  mixture  is  coagulated  at  70a  C.  in  slanted  tubes  and  sterilized  at 
low  temperatures  by  the  fractional  method.  Upon  this  medium  'the 
diphtheria  bacillus  in  twelve  to  twenty-four  hours  develops  minute, 
grayish-white,  glistening  colonies.  These  enlarge  rapidly,  soon  out- 
stripping the  usually  accompanying  streptococci.  The  medium  seems 
to  possess  almost  selective  powers  for  the  bacillus  and,  for  this  reason, 
it  is  especially  valuable  for  diagnostic  purposes. 

Meat-infusion  Agar. — Upon  slightly  alkalin  meat-infusion  agar  the 
bacillus  develops  readily,  though  less  so  than  on  Loeffler 's  serum. 
Organisms  which  have  been  on  artificial  media  for  one  or  more  genera- 
tions may  grow  with  speed  and  luxuriance  upon  this  medium.  When 
planted  directly  from  the  human  or  animal  body  upon  agar,  however, 
growth  may  occasionally  be  slow  and  extremely  delicate.  Colonies  on 
agar  appear  within  twenty-four  to  thirty-six  hours  as  small,  rather 
translucent,  grayish  specks.  The  appearance  of  these  colonies  is  quite 
characteristic  and  easily  recognized  by  the  practiced  observer.  Surface 
colonies  are  irregularly  round  or  oval,  showing  a  dark,  heaped-up, 
nucleus-like  center,  fringed  about  by  a  loose,  coarsely  granular  disk. 
The  edges  have  a  peculiarly  irregular,  torn  appearance  which  distin- 
guishes them  readily  from  the  sharply  defined  streptococcus  colonies. 

10  Michel,  Cent.  f.  Bakt.,  1897. 


566 


PATHOGENIC  MICROORGANISMS 


For  these  reasons  agar  is  the  medium  most  commonly  used  for  purposes 
of  isolation. 

The  addition  of  dextrose  1  per  cent,  nutrose  2  per  cent,  or  glycerine 
6  per  cent,  renders  agar  more  favrable  for  rapid  growth,  but  unfits  it 
for  the  preservation  of  cultures,  the  organism  dying  out  more  rapidly, 

probably  because  of  acid  forma- 
tion. 

Meat-infusion  Broth .  —  Upon 
beef  or  veal  broth  the  diphtheria 
bacillus  grows  rapidly,  almost  in- 
variably forming  a  pellicle  upon 
the  surface, — another  expression 
of  its  desire  for  oxygen.  The  broth 
remains  clear.  Broth  tubes  with 
such  growth,  therefore,  have  a 
characteristic  appearance. 

Meat-infusion  gelatin  is  a  favor- 
able  medium  for  the  Klebs-Loeffler 
bacillus,   but  growth  takes  place 
slowly  because  of  the  low  temper- 
FIG.  60 —COLONIES  OF  DIPHTHERIA      ature  at  which  this  medium  must 
ON  GLYCERINE  AGAR.  be  kept.     Gelatin  is  not  fluidified. 

Milk   is   an  excellent  medium, 

and  for  this  reason  may  even  occasionally  be  a  vehicle  of  transmis- 
sion.    There  is  no  coagulation  of  the  milk. 

Upon  potato,  B.  diphtherise  will  grow  only  after  neutralization  of  the 
acid.  It  is,  at  best,  however,  a  poor  nutrient  medium. 

Upon  the  various  pepton  solutions  the  bacillus  of  diphtheria  produces 
no  indol. 

Many  special  media  have  been  recommended  for  the  cultivation  of 
this  organism.  The  most  important  of  these  are  the  modification  of 
Loeffler's  serum  devised  by  Beck,11  the  horse-blood-fibrin  cake  used  by 
Escherich,  and  Wassermann's  ascitic-fluid-nutrose-agar,  called  by  him 
"Nasgar."  None  of  these  has  sufficient  advantages  over  the  simpler 
media,  however,  to  make  its  substitution  desirable. 

Isolation. — Cultures  are  taken  from  throats  upon  Loeffler's  blood 
serum.  These  are  permitted  to  grow  at  37.5°  C.  for  from  eighteen  to 
twenty-four  hours.  At  the  end  of  this  time  about  5  c.c.  of  bouillon 


11 M.  Beclc,  Kolle  uiul  Wasscrmann;  Brit.  Mettl.  Jour. 


BACILLUS  DIPHTHERIA  567 

are  poured  into  the  tubes  and  the  growth  is  gently  emulsified  in  the 
broth  with  a  platinum  loop.  Two  or  three  loopfuls  of  this  emulsion 
are  then  streaked  over  the  surface  of  glucose  agar,  serum  agar,  or  nutrose 
agar.  After  twenty-four  hours'  incubation  these  plates  show  charac- 
teristic colonies  which  can  be  easily  fished  and  again  transferred  to 
Loeffler  tubes  or  any  other  suitable  medium. 

Diagnosis. — Cultures  from  suspected  throats  are  taken  on 
Loeffler's  blood  serum  medium  and  incubated  at  37.5°  C.  for  12  to  18 
hours.  At  the  end  of  this  time  morphological  examination  by  stain- 
ing with  Loeffler's  alkalin  methylene  blue  and  by  some  polar  body  stain 
like  that  of  Neisser  is  carried  out.  Occasionally  direct  smears  from  the 
throat  may  show  the  bacilli,  but  it  is  rarely  possible  to  make  a  satis- 
factory diagnosis  in  this  way. 

Williams  has  pointed  out  that  in  throat  cultures  in  which  the  diph- 
theria bacilli  are  few  in  number  it  is  of  advantage  to  inoculate  a  tube  of 
ascitic  broth  with  the  mixed  culture.  The  diphtheria  bacilli  will  appear 
in  eighteen  to  twenty-four  hours  as  a  pellicle  on  the  surface.  A  por- 
tion of  this  pellicle  may  then  be  plated  on  ascitic  agar  and  isolated  in 
pure  culture  from  the  colonies.  This,  however,  is  not  necessary  for 
routine  examinations.  The  important  point  is  to  take  cultures  as  early 
as  possible  on  fresh  and  moist  Loeffler's  medium,  avoiding  the  dried 
tubes  so  often  passed  out  from  old  stock  at  drug  store  stations.  It  is 
important  to  smear  from  the  actually  involved  areas,  examining  the 
throat  with  good  illumination,  and  to  have  the  tubes  incubated  without 
delay,  instead  of  carrying  them  about  for  hours  after  inoculation. 
Such  cultures,  examined  by  an  experienced  man  should  give  positive 
diagnoses  in  almost  all  of  the  actual  cases.  In  the  diagnosis  of  children 
and  in  carrier  work  it  is  important  to  take  nasal  as  well  as  throat  cul- 
tures. 

Pathogenicity. — Bacillus  diphtherias  causes  a  more  or  less  specific 
local  reaction  in  mucous  membranes,  which  results  in  the  formation  of 
the  so-called  "  pseudo-membranes."  When  these  are  characteristically 
present,  infection  with  this  bacillus  should  always  be  suspected.  It 
should  be  remembered,  however,  that  membranous  inflammation  is  not 
necessarily  present  in  all  cases.  We  have  seen  positive  cultures  in  a 
considerable  number  of  people,  especially  in  relatively  insusceptible 
individuals,  in  whom  the  throat  showed  nothing  more  than  severe  con- 
gestion and  catarrhal  inflammation.  The  consequent  disease  depends, 
in  part,  upon  the  mechanical  disturbance  caused  by  the  local  inflamma- 
tion and,  in  part,  upon  the  systemic  poisoning  with  the  toxin  which  the 


568  PATHOGENIC   MICROORGANISMS 

bacilli  produce.  Although  the  diphtheria  bacillus  has  been  found  after 
death  in  the  spleen  and  liver,  we  have  no  data  which  would  justify  the 
assumption  that  a  true  diphtheria-septicemia  may  occur  during  life. 
It  is  probable  that  in  those  cases  which  Baginsky  12  has  called  the  sep- 
ticemic  form  of  diphtheria,  bacillus  diphtheria  has  merely  opened  a 
path  by  which  accompanying  streptococci  have  gained  access  to  the 
lymphatics  and  the  blood  stream.  The  most  frequent  sites  of  diph- 
theritic inflammations  are  the  mucous  membranes  of  the  throat,  larynx, 
and  nose.  They  have  also  been  found  in  the  ear,  upon  the  mucous 
membrane  of  the  stomach  and  the  vulva,  and  upon  the  conjunctiva  and 
the  skin.  According  to  Loeffler,  Strelitz,13  and  others,  the  bacillus  may, 
by  extension  from  the  larynx,  give  rise  to  a  true  diphtheritic  broncho- 
pneumonia. 

Thus,  the  localized  injury  due  to  the  very  violent  inflammatory 
reaction  elicited  by  the  diptheria  bacilli  at  their  point  of  lodgment  which, 
in  the  large  majority  of  cases,  is  in  the  upper  respiratory  tract,  especially 
pharynx  and  tonsils,  are  the  immediately  noticeable  and  visible  changes 
in  the  disease  of  human  beings.  In  attempting  to  make  a  diagnosis  of 
these  by  inspection,  the  clinician  should  remember  that  the  pseudo- 
membranous  inflammation  characterized  by  its  adherence  to  the 
submucosa  and  the  bleeding  points  it  leaves  on  being  scrapped,  is  not 
pathognomonic  of  the  diphtheria  bacilli,  but  means  simply  a  very 
violent  inflammatory  reaction  and  that,  while  this  condition  is  most 
commonly  caused  by  the  diphtheria  bacillis,  a  great  many  other  severe 
injuries  or  inflammations  may  give  rise  to  very  similar  appearances. 
Thus,  a  very  severe  streptococcus  infection  of  the  throat  may  simulate  a 
diphtheritic  membrane  and  escharotics  or  other  chemical  or  mechanical 
injuries  may  give  rise  to  simpler  lesions.  Another  point  of  considerable 
clinical  importance  is  the  fact  that  diphtheritic  inflammation  of  the 
throat  may  often  be  associated  with  other  concomitant  infectious 
processes.  Streptococcus  infections  superimposed  upon  a  local  diph- 
theria infection,  somewhat  changes  the  appearance,  both  of  the  local 
lesion  and  of  the  general  clinical  picture  and  is  apt  to  lead  to  a  very  much 
greater  severity  of  the  illness.  Another  common  experience  is  to  find, 
at  the  site  of  the  inflammation,  an  ulcerative  process,  smears  from  which 
on  staining  with  Gram's  gential  violet  or  carbolfuchsin  will  show  a 
typical  picture  of  Vincent/s  angina  with  the  fusiform  bacilli  and  the 
spirilla  characteristic  of  this  infection.  Owing  to  a  number  of  mis- 

12  Baginsky,  "Lehrbueb.  d.  Kinderkrankheiteri." 
"Strelits,  Arch.  f.  Kimlerheilk.,  1891 


BACILLUS  DIPHTHERIA  569 

takes  we  have  made,  it  has  become  our  rule  whenever  we  see  a  case  of 
Vincent's  angina  to  take  cultures  on  Loeffer's  medium  as  for  diphtheria 
diagnosis,  since  we  have  on  two  or  three  occasions  found  almost  pure 
cultures  of  diphtheria  bacilli  taken  from  the  depths  of  such  anginal 
ulcers.  The  systemic  symptoms  in  diphtheria  are  not  always  severe 
since,  as  we  shall  see,  adults  are  apt  to  be  particlly  protected  by  the 
presence  of  diphtheria  antitoxin  in  their  blood  and  in  consequence  their 
disease  may  be  both  locally  and  systemically  so  mild  that  diphtheria 
might  not  be  seriously  considered  on  purely  clinical  evidence. 

The  mechanical  injury  may  actually  lead  to  death.  This  is  par- 
ticularly due  in  the  care  of  children  in  whom  extension  downward  with 
membrane  formation  in  the  larynx  may  lead  to  laryngeal  obstruction, 
necessitating  tracheotomy  or  intertubation.  While  layrngeal  obstruc- 
tion was  often  the  cause  of  death  in  children  in  former  years,  it  has 
fortunately  been  rendered  much  less  frequent  by  antitoxin  treatment, 
by  the  greater  vigilance  of  doctors  and  their  ability  to  make  an  early 
diagnosis.  The  responsibility  of  the  physician  in  this  regard  is  a  grave 
one,  since  early  diagnosis  and  consequent  antitoxin  treatment  is  the 
only  way  to  prevent  such  accidents. 

The  general  symptoms  of  diphtheria  are  due  to  the  action  of  the 
toxin  absorbed  from  the  lesion.  These  consist  in  increased  temperature, 
rapid  pulse,  headache,  muscular  pains,  etc.  In  severe  infections  there 
may  be  erythematous  eruptions.  Death  in  diphtheria  may  occur  as  a 
consequence  of  secondary  bronco-pneumonia,  but  in  uncomplicated 
cases  is  usually  attributed  to  circulatory  failure.  According  to  experi- 
ments done  by  MacCullum  14  in  the  perfusion  of  the  heart  with  diph- 
theria toxin,  the  toxin  does  not  seem  to  act  directly  upon  the  heart,  in 
spite  of  the  fact  that  there  seems  to  be  definite  evidence  that  the  heart  is 
involved  in  diphtheria.  According  to  the  same  investigator,  there  dose 
not  seem  to  be  sufficient  gross  or  microscopic  changes  in  the  hearts  of 
people  dead  of  diphtheria  to  explain  death.  It  is  MacCallum  opinion's 
that  Passler  and  Romberg  were  probably  right  in  stating  that  the  effect 
of  the  poison  is  "  chiefly  upon  the  vasomotor  control  of  the  blood  ves- 
sels." Injuries  to  other  organs  are  apparent  in  albuminuria  due  to 
acute  interstitial  nephritis,  and  cloudy  swelling  of  parenchyma  cells  in 
other  organs.  In  view  of  the  marked  changes  in  the  suprarenal  bodies 
in  guinea  pigs  treated  with  diphtheria  toxin,  these  organs  have  been 
especially  investigated  in  diphtheria,  and  though  usually  little  or  no 


14  MacCallum,  Textbook  of  Pathology. 


570  PATHOGENIC   MICROORGANISMS 

change  has  been  found,  MacCallum  states  that  "the  changes  in  the 
adrenals  are  likely  to  be  more  intense  than  in  most  of  the  other  organs, " 
showing  hemorrhages  and  cellular  degeneration.  This  may  have  direct 
bearing  on  the  abnormal  fall  in  blood  pressure.  There  is  a  marked 
action  on  the  part  of  the  diphtheria  toxin  upon  nerve  tissues,  both 
neurons  and  cells.  This  results  in  post-diphtheritic  paralyses  which 
occur  particularly  in  cases  that  have  been  insufficiently  or  not  at  all 
treated  with  antitoxin.  The  paralyses  are  particularly  frequent  in  the 
areas  supplied  by  the  cranial  nerves  and  among  the  most  common  forms 
are  paralysis  of  the  muscles  of  the  soft  palate  of  the  larynx  and  the  ocular 
muscles.  Paralysis,  however,  may  also  appear  in  other  parts  of  the 
body.  We  have  seen  several  cases  where  a  sore  throat  was  not  sus- 
pected of  being  diphtheria  until  late  paralysis  had  occurred. 

For  the  usual  laboratory  animals  the  diphtheria  bacillus  is  highly 
pathogenic.  Dogs,  cats,  fowl,  rabbits,  and  guinea-pigs  are  susceptible. 
Rats  and  mice  are  resistant.  False  membranes,  analogous  to  those 
found  in  human  beings,  have  been  produced  in  many  animals,  but  only 
when  inoculation  had  been  preceded  by  mechanical  injury  of  the 
mucosa.  Small  quantities  (0.5  to  1  c.c.)  of  a  virulent  broth  culture, 
given  subcutaneously  to  a  guinea-pig,  may  produce  the  greatest  symp- 
toms and  within  six  to  eight  hours  the  animal  may  show  signs  of  great 
discomfort.  Death  occurs  usually  within  thirty-six  to  seventy-two 
hours.  Upon  autopsy  the  point  of  inoculation  is  soggy  and  serosan- 
guineous  exudate;  neighboring  lymph-nodes  are  edematous.  Lungs, 
liver,  spleen,  and  kidneys  are  congested.  There  may  be  pleuritic  and 
peritoneal  exudates.  Pathognomonic  is  a  severe  congestion  of  both 
suprarenal  bodies.  The  gastric  ulceration  recently  described  by  Rose- 
nau  and  Anderson  15  may  occur,  but  are  by  no  means  regularly  found 
(two  out  of  fifty  in  our  series).16 

Determination  of  Virulence. — When  diphtheria  or  diphtheria-like 
bacilli  are  isolated  from  the  throats  of  patients  not  showing  typical 
clinical  diphtheria,  or  from  healthy  individuals  suspected  of  being 
carriers,  it  is  important  to  determine  whether  these  organisms  are 
toxin  producers.  The  usual  criterion  is  their  virulence  for  guinea-pigs. 
Two  c.c.  of  a  forty-eight-hour  broth  or  ascitic  broth  culture  are  injected 
subcutaneously  into  a  normal  guinea-pig.  This  dose  will  kill  the  pig  in 
three  to  five  days  if  the  culture  is  virulent.  A  control  injection  should 


ln  Eoscnau  and  Anderson,  Jonr.  Inf.  Bis.,  iv,  1907. 
16  Zinsser,  Jonrn.  Mod.  Res.,  xvii,  1907. 


BACILLUS  DIPHTHERIA  571 

always  be  made  into  another  pig  of  the  same  weight,  which  has  received 
an  injection  of  antitoxin  (at  least  250  units)  twelve  to  twenty-four  hours 
previously.  Recently  Neisser  has  suggested  that  the  intracutaneous 
injection  of  the  suspected  bacilli  may  be  used  for  the  determination  of 
virulence.  This  has  the  advantage  of  economy,  as  several  tests  can  be 
carried  out  on  the  same  pig.  The  method  as  applied  by  Zingher  and 
Soletsky  17  has  been  to  use  the  following  modification  of  Neisser's 
method:  Two  guinea-pigs  of  about  250  gr.  are  used  for  the  test.  The 
abdominal  wall  is  prepared  by  shaving  or  plucking  out  the  hair.  A 
twenty-four-hour  pure  culture  on  Loeffler's  medium  is  emulsified  in 
20  c.c.  of  normal  salt  solution  and  0.15  c.c.  of  this  suspension  is  injected 
intracutaneously  at  a  corresponding  site  into  each  of  the  two  guinea- 
pigs.  One  of  these  animals  is  given  at  the  same  time  an  intracardial 
injection  of  about  250  units  of  antitoxin,  or  preferably  is  prepared  by  an 
intraperitoneal  injection  of  antitoxin  twenty-four  hours  before  the  tests 
are  made.  Six  cultures  may  be  tested  in  this  way  on  two  animals. 
Virulent  strains  produce  a  definitely  circumscribed  local  infiltrated 
lesion,  which  shows  superficial  necrosis  in  two  to  three  days.  In  the 
control  pig  the  skin  remains  normal.  This  method,  in  the  hands  of 
Zingher,  gives  results  parallel  to  those  obtained  with  the  subcutaneous 
tests. 

Diphtheria  Toxin.18 — Animals  and  man  infected  with  B.  diph- 
therise  show  evidences  of  severe  systemic  disturbances  and  even  organic 
degenerations,  while  the  microorganism  itself  can  be  found  in  the  local 
lesion  only.  This  fact  led  even  the  earliest  observers  to  suspect  that, 
in  part  at  least,  the  harmful  results  of  such  an  infection  were  attrib- 
utable to  a  soluble  and  diffusible  poison  elaborated  by  the  bacillus. 
The  actual  existence  of  such  a  poison  or  toxin  was  definitely  proved  by 
Roux  and  Yersin19  in  1889.  They  demonstrated  that  broth  cultures 
in  which  B.  diphtheria  had  been  grown  for  varying  periods  would 
remain  toxic  for  guinea-pigs  after  the  organisms  themselves  had  been 
removed  from  the  culture  fluid  by  filtration  through  a  Chamberland 
filter. 

METHODS  OF  PRODUCTION  OF  DIPHTHERIA  TOXIN. — While  toxin  can 
be  produced  with  almost  all  of  the  virulent  diphtheria  bacilli,  there  is 
great  variation  in  the  speed  and  degree  of  production,  dependent  upon 
the  strain  of  organisms  employed  and  upon  the  ingredients  and  reac- 

17  Zingher  and  Soletsky,  Jour.  Inf.  Dis.,  1916,  xvii,  54. 
™Loefller,  Cent.  f.  Bakt.,  1887. 
19  Roux  and  Yersin,  loc.  cti. 


572  PATHOGENIC  MICROORGANISMS 

tion  of  the  medium  upon  which  they  are  grown.  Most  laboratories 
possess  one  or  several  strains  of  bacilli  which  are  empirically  known 
to  be  especially  potent  in  this  respect.  One  of  the  most  extensively 
used,  not  only  in  this  country  but  in  Europe  as  well,  is  the  strain  known 
as  "Culture  Americana,"  or  "Park- Williams  Bacillus  No.  8,"  an 
organism  isolated  by  Dr.  Anna  Williams  of  the  New  York  Department 
of  Health  in  1894.  Throughout  more  than  ten  years  of  cultivation 
this  bacillus  has  retained  its  great  power  of  toxin  production. 

Because  of  the  severity  of  cases  of  diphtheria  in  which  the  diphtheria 
bacilli  were  associated  with  streptococci,  many  observers  were  led  to 
believe  that  the  presence  of  streptococci  tended  to  increase  the  toxin- 
producing  power  of  B.  diphtheria.  Experiments  by  Hilbert,20  Theobald 
Smith,21  and  others  seem  to  have  given  support  to  this  view. 

The  medium  most  frequently  employed  for  the  production  of  toxin 
is  a  beef-infusion  broth.  There  are  minor  differences  of  opinion  as  to 
the  most  favorable  constitution  of  this  medium  for  the  production  of 
toxin.  All  agree,  however,  in  recognizing  the  importance  of  pepton, 
without  which,  according  to  Madsen,22  no  satisfactory  toxin  has  yet 
been  produced.  This  is  added  in  proportions  of  from  1  to  2  per  cent. 
The  presence  of  sugars  in  the  medium  is  not  desirable  in  that  it  leads  to 
acid  production;  L.  Martin23  removes  the  sugars  from  the  meat  by 
fermentation  with  yeast.  Smith  24  accomplishes  the  same  purpose  with 
B.  coli.  According  to  Park  and  Williams,25  however,  this  is  super- 
fluous, the  quantity  of  sugar  present  in  ordinary  butcher's  meat  not 
being  sufficient  to  exert  unfavorable  influence. 

Experience  has  shown  that  a  primary  alkalin  reaction  offers  the 
most  favorable  conditions  for  toxin  production.  In  all  cultures  of  B. 
diphtherias  in  non-sugar  free  broth,  there  is,  at  first,  a  production  of 
acid  and,  while  this  continues,  there  is,  as  Spronk26  has  shown,  little  or 
no  evidence  of  toxin  elaboration.  Park  and  Williams,27  in  an  inquiry 
into  the  question  of  reaction,  came  to  the  conclusion  that  the  best 
results  are  obtained  with  a  broth  to  which,  after  neutralization  to 


20  Hilbert,  Zeit.  f .  Hyg.,  xxix,  1898. 

21  Smith,  Medical  Rec.,  May,  1896. 

22  Madsen,  Krausund  Levaditi,  "Haiidbuch  d.  Technic,"  etc.,  1907. 

23  L.  Martin,  Ann.  de  Pinst.  Pasteur,  1897. 

24  Th.  Smith,  Jour.  Exp.  Med.,  iv,  1899. 
25Parfc  and  Williams,  Jour.  Exp.  Med.,  1897. 
28  Spronk,  Ann.  de  1  'inst.  Pasteur,  1895. 

27  Parlc  and  Williams,  Jour.  Exp.  Med.,  1897. 


BACILLUS  DIPHTHERIA  573 

litmus,  N/l  NaOH  is  added  in  an  amount  of  7  c.c.  to  the  liter.  In  such 
a  medium  the  largest  yield  of  toxin  is  obtained  after  about  five  to  eight 
days'  growth  at  a  temperature  of  37.5°  C. 

Free  access  of  oxygen  to  the  culture  medium  during  the  growth  of 
the  organisms  has  been  found  to  be  of  great  importance.  Roux  obtained 
this  by  passing  a  stream  of  oxygen  through  the  bouillon.  The  supply 
is  quite  sufficient  for  practical  purposes,  however,  if  the  medium  is 
distributed  in  thin  layers  in  large-necked  Erlenmeyer  flasks. 

CHEMICAL  NATURE  AND  PHYSICAL  PROPERTIES  OF  DIPHTHERIA 
TOXIN. — The  chemical  composition  of  diphtheria  toxin  is  not  known. 
Brieger  and  Frankel,28  by  repeated  precipitation  with  alcohol,  suc- 
ceeded in  extracting  from  toxic  bouillon  a  white,  water-soluble  powder 
which  possessed  most  of  the  poisonous  properties  of  the  broth  itself. 
This,  in  solution,  gave  many  of  the  useful  protein  reactions,  but  dif- 
fered from  proteins  in  failing  to  coagulate  when  boiled  and  in  not  giving 
precipitates  when  treated  with  magnesium  sulphate,  sodium  sulphate, 
or  nitric  acid.  It  was  believed  by  them  to  be  closely  related  to  the 
albumoses,  bodies  representing  intermediate  phases  in  the  peptoniza- 
tion  of  albumins.  Similar  results  have  been  obtained  by  Wassermann 
and  Proskauer,29  Brieger  and  Boer,30  and  others.  Uschinsky,31  on 
the  other  hand,  has  disputed  the  protein  nature  of  toxins  in  general, 
having  produced  diphtheria  toxin  by  growing  the  organism  upon  a 
medium  entirely  free  from  albuminous  bodies.  Uschinsky  believes 
that  the  protein  reactions  observed  by  others  may  be  due  to  ingredients 
of  the  precipitates  other  than  the  toxin.  It  is  not  impossible,  however, 
that  the  organisms  may  have  produced  protein  substances  by  synthesis 
from  the  simpler  substances  in  Uschinsky's  medium.  The  production 
of  toxin  from  such  a  medium,  therefore,  is  not  a  conclusive  argument 
against  the  protein  nature  of  toxins.  Accurate  chemical  isolation  and 
analysis  of  diphtheria  toxin  have  not  yet  been  accomplished. 

Diphtheria  toxin  is  destroyed,32  when  in  the  fluid  form,  by  tempera- 
tures of  58°  to  60°  C.  In  the  dry  state,  it  resists  a  temperature  of  70°  C. 
and  over,  without  change.  Light  and  free  access  of  air  produce  rapid 
deterioration.  Sealed,  protected  from  light,  and  kept  at  almost  freezing 


28  Brieger  imd  Frankel,  Berl.  klin.  Woch.,  xi_xii,  1889. 

-'•'  \\' asxcrmann  nnd   /Vo.s7,Y////',  Dent.  med.  Woch.,   1891,  p.  585. 

3(1  Itricyer  nnd  Boer,  Dcut.  mod.  Woch.,  1896,  p.  783. 

31  UschinsTcy,  Cent,  f .  Bakt.,  xxi,  1897. 

82  Roux  et  Yersin,  loc.  cit. 


574  PATHOGENIC   MICROORGANISMS 

point,  the  toxin  remains  stable  for  long  periods.      Electrical   currents 
passed  through  toxic  broth  have  little  or  no  effect  upon  it. 

Epidemiology  of  Diphtheria.33 — There  is  no  disease  in  which 
sanitary  control  can  accomplish  so  much  as  in  diphtheria  because  we 
are  furnished  in  this  instance  not  only  with  accurate  and  simple  methods 
of  bacteriological  diagnosis,  of  cases  and  carriers,  but  we  have  available 
a  specific  susceptibility  test  by  means  of  which  we  can  pick  out  the 
susceptibles  in  any  group,  as  well  as  methods  of  prophylactic  protection 
which  allow  a  choice  between  a  speedy  and  a  slow  procedure,  both  of 
them  of  proven  value.  In  this  disease,  sanitary  measures  have  made 
tremendous  strides  and  greatly  reduced  both  morbidity  and  death 
rate  since  the  introduction  of  bacteriological  methods.  Newsholm34 
has  studied  death  rates  in  diphtheria,  and  found  that  in  earlier  years, 
great  epidemics  of  diphtheria  used  to  spread  through  the  great  cities. 
There  was  such  an  epidemic  in  London  in  1874.  In  1872  he  states  that 
the  death  rate  from  diphtheria  and  croup  in  Boston  was  35  per  100,000, 
but  in  1881,  it  was  close  to  218  per  100,000.  Epidemic  waves  seem  to 
have  recurred  at  five-  and  ten-year  intervals,  but  even  in  the  inter- 
epidemic  years  in  cities  generally,  the  death  rate  seems  to  have  ranged 
anywhere  from  20  to  60  per  100,000.  It  was,  and  is  always  endemic 
all  over  the  world,  is  somewhat  more  prevalent  in  colder  climates  where 
upper  respiratory  inflammations  are  more  common,  and,  for  some 
unknown  reason,  has  been  relatively  more  common  in  rural  than  in 
urban  communities.  Quite  naturally,  the  disease  has  always  been 
particularly  a  school  disease  among  children.  From  studies  by  Schick,35 
Hahn,36  and  others,  it  appears  that  the  new-born  child  is  endowed  with  a 
certain  amount  of  diphtheria  antitoxin  by  the  mother,  probably  through 
the  placenta,  to  some  degree  perhaps  transmitted  through  the  colos- 
trum. This  relative  immunity  fades  at  the  end  of  the  first  year  of  life 
and  is  gradually  reacquired  so  that  the  most  susceptible  years  are 
between  one  and  perhaps  nine  or  ten  years  of  age.  In  the  army,  with 
an  age  group  ranging  from  twenty  to  thirty  years,  it  was  found,  during 
the  recent  war,  that  about  10  per  cent  of  the  personnel  was  Schick 
positive  or,  therefore,  susceptible.  For  more  accurate  susceptibility 
statistics  we  must  await  more  extensive  measurements  and  statistical 
studies  of  Schick  tests. 

33  See  Zinsser,  Nelson  ;s  Loose-Leaf  Medicine,  9,  205. 
™  Newsholm,  Epidemic  Diphtheria,  London,  1898. 
35  ScMck,  Ueber  Diphtherimmunitat.,  Wiesbaden,  1910. 
"Hahn,  Dent,  mcd.  Woch.,  38,  1912,  1366. 


BACILLUS   DIPHTHERIA  575 

The  striking  distance  of  diphtheria  is  not  very  large,  but  the  organism 
is  relatively  resistant  to  the  ordinary  influences  of  exterior  corporal 
circumstances,  and  may  live  for  considerable  lengths  of  time  in  mucus 
or  saliva  deposited  on  heating  utensils,  playthings,  pencils,  handker- 
chiefs, etc.  In  dried  bits  of  membrane  the  bacilli  may  live  for  many 
weeks  as  Loeffler  has  shown. 

Diphtheria  is  transmitted  from  one  individual  to  another  directly 
or  indirectly  by  contact  or  droplet  infection — as  in  coughing,  etc.  It- 
has  been  found  that  individuals  may  retain  virulent  diphtheria  bacilli 
in  nose  and  throat  for  long  periods  after  recovery  from  the  disease. 
These  are  the  so-called  " diphtheria  carriers." 

The  problem  of  diphtheria  carriers  has  become  one  of  considerable 
importance  and  has  been  given  special  prominence  of  recent  years  by 
the  studies  of  Von  Scholly,  Moss,  and  Nuttall  and  Graham  Smith. 
Anderson,  Goldberger  and  Hachtel37  studied  4,039  healthy  people  in 
the  city  of  Detroit,  and  found  that  0.928  per  cent  harbored  bacilli 
identical  morphologically  with  the  Klebs-Loeffler  bacillus.  This  figure 
is  rather  lower  than  those  of  some  other  investigators,  but  would  indi- 
cate, as  the  writers  stated,  that  there  were  from  5000  to  6000  diphtheria 
carriers  in  the  city  of  Detroit. 

Of  19  cultures  isolated  from  19  of  the  carriers,  only  2  were  virulent, 
which  would  indicate  that  only  0.097  per  cent  of  the  people  examined 
carried  organisms  capable  of  producing  disease.  An  interesting  further 
point  is  that  the  bacillus  Hoffmanni  was  present  in  at  least  41.9  per  cent 
of  over  2000  individuals  examined,  and  that  47  cultures,  morphologically 
identified  as  Bacillus  Hoffmanni,  were  avirulent.  This  would  confirm 
the  impression  gained,  we  believe,  by  most  experienced  laboratory 
workers  that  a  true  Hoffmanni  can  be  distinguished  with  considerable 
certainty  from  a  Klebs-Loeffler  bacillus  by  morphological  examination 
alone,  and  that  its  significance  is  probably  that  of  a  frequently  present 
saprophyte  of  the  throat  and  pharynx.  The  studies  of  Goldberger, 
Williams  and  Hachtel  also  indicate  that  in  examining  for  diphtheria 
carriers  it  is  best  not  to  confine  oneself  either  to  the  nose  or  throat,  but 
that  cultures  should  be  taken  from  both  places  in  every  case. 

Carriers  naturally  increase  in  crowded  communities  in  the  course  of 
cold  weather,  when  nasopharyngeal  catarrhs  are  common,  and,  we  have 
seen  as  high  as  17  per  cent  carriers  in  a  military  unit  in  which  diphtheria 


37  Goldberger,   Williams  and  Haclitel,  Bull.  No.   101,  of  the  Hygienic  Labora- 
tories, of  the  IT.  S.  Public  Health  Service. 


57C)  PATHOGENIC   MICROORGANISMS 

and  other  respiratory  diseases  were  prevalent  soon  after  they  had  to  be 
crowded  together  during  Transatlantic  transportation.  The  carrier 
state  in  kitchen  personnel  is  particularly  important,  and  attention 
should  be  paid  to  these  groups  in  a  community  whenever  the  source  of 
infection  is  being  traced.  Indirect  and  direct  contact  with  carriers 
is  probably  the  most  common  method  by  which  the  disease  is  kept 
going  in  modern  communities.  Less  important,  though  still  of  some 
significance,  is  food  transmission  and  milk  epidemics  have  not  been 
uncommon.  Also,  these  will  mean  infection  of  the  milk  by  a  milk 
handler,  who  is  a  carrier  or  suffering  from  a  mild  diphtheria.  How- 
ever, in  one  instance  diphtheria  bacilli  were  isolated  from  the  inflamed 
udders  of  a  cow. 

The  carrier  problem  thus  becomes  the  most  important  epidemic- 
logical  feature.  The  carriers  may  either  be  temporary  or  chronic. 
Convalescent  diphtheria  cases  usually  get  rid  of  their  bacilli  sponta- 
neously within  two,  three,  or  four  weeks.  Healthy  individuals  exposed 
to  cases  or  carriers  as  a  rule  do  not  keep  their  organisms  more  than 
either  a  few  days  or  a  few  weeks,  depending  perhaps  to  some  extent 
upon  the  condition  of  the  mucous  membranes.  A  small  percentage 
remain  chronic  carriers  and,  in  isolated  instances,  it  has  seemed  almost 
impossible  to  free  these  individuals  of  their  diphtheria  bacilli.  They 
are,  however,  such  definite  menaces  that  prolonged  isolation  and  vigor- 
ous attempts  at  cure  must  be  made  for  the  protection  of  the  com- 
munity. In  all  such  cases,  virulence  tests  should  be  made,  since  pro- 
longed isolation  implies  so  much  interference  with  the  life  of  the  affected 
individual  that  it  would  be  quite  improper  to  confine  a  person  unless 
we  were  sure  that  the  organisms  carried  by  him  were  capable  of  trans- 
mitting the  disease. 

Many  different  methods  have  been  employed  for  the  freeing  of 
carriers.  Not  a  single  one  of  them,  however,  has  been  permanently 
successful.  Practically  all  the  ordinary  throat  antiseptics,  peroxide 
of  hydrogen,  permanganate  of  potassium,  iodin  and  glycerin,  weak 
formaldehyd,  various  hypochlorite  solutions  have  been  used,  some  of 
them  with  occasional  success,  but  without  regularity  in  any  of  them. 
Acriflavine  and  other  dyes  have  been  applied  and  implantation  of 
staphylococci  or  some  of  the  acidophilic  bacteria  upon  the  throat  in  the 
hope  of  driving  out  the  diphtheria  bacilli  by  bacterial  competition  have 
been  tried  and  have  usually  failed.  The  spraying  of  the  throat  with 
pyocyanase  was  in  vogue  a  few  years  ago,  but  cannot  be  said  to  have 
brought  encouraging  results.  It  is  probable  that  quite  the  most  impor- 


BACILLUS  DIPHTHERIA  577 

tant  thing  is  to  correct  pathological  conditions  of  the  nose  and  throat  by 
the  correction  of  a  deviated  septum  or  the  removal  of  tonsils  and  ade- 
noids, whenever  necessary.  Added  to  this,  sunshine,  cleanliness  and 
the  cure  of  chronic  catarrhal  inflammation  are  probably  more  important 
than  any  kind  of  antiseptic  treatment. 

The  detection  of  carriers  can  easily  be  carried  out  on  a  large  scale 
by  a  relatively  small  force  of  bacteriologists.  It  requires  a  large  supply 
of  sterile  swabs  and  Loeffler's  medium,  and  wholesale  cultures  can  be 
taken  on  a  group  as  large  as  entire  infantry  regiments  at  war  strength 
without  too  great  an  expenditure  of  energy. 

The  prevention  of  diphtheria  for  all  these  reasons  falls  into  a  very 
logical  system  of  procedure.  Whenever  diphtheria  breaks  out  in  a 
school,  institution,  military  unit,  factory  unit,  etc.,  etc.,  the  first  thing 
to  do  is  to  make  a  thorough  inspection  and  immediately  institute  pre- 
cautions against  the  transmission  of  mucus  from  one  individual  to 
another.  This  will  imply  supervision  of  the  kitchen,  food  preparation, 
dish  washing,  prohibition  of  spitting,  isolation  of  individuals  who  are 
coughing  and  hawking  or  suffering  from  severe  catarrhal  inflammations; 
cleanliness  of  sleeping  quarters  and  mess  halls,  inspection  of  the  entire 
group,  and  the  taking  of  throat  cultures;  segregation  of  those  that  show 
positive  cultures;  especially  attention  in  this  regard,  and  repeated 
culturing  of  kitchen  personnel  and  food  handlers ;  Schick  reactions  upon 
the  entire  group,  with  prophylactic  immunization  (preferably  by  the 
active  method),  of  those  with  positive  Schick  reactions;  subsequent 
attention  to  chronic  carriers.  These  precautions  are  simple  and 
applicable  with  minor  modifications  to  any  kind  of  group  in  which 
diphtheria  appears. 

Shick  Reaction. — The  studies  of  Roemer  and  others  have  shown 
that  the  blood  of  the  majority  of  normal  adults  contains  a  small  amount 
of  diphtheria  antitoxin.  This  normal  antitoxin  probably  accounts  for 
resistance  of  many  individuals  to  diphtheria.  Its  presence  may  be 
very  easily  detected  by  means  of  the  Schick  reaction.  A  standardized 
diphtheria  toxin  is  diluted  in  normal  saline  so  that  0.1  c.c.  of  the  solu- 
contains  Vso  M.L.D.  for  a  guinea-pig.  This  amount  is  injected  intra- 
cutaneously.  If  the  blood  of  the  subject  has  less  than  1/30  unit  of 
antitoxin  per  cubic  centimeter,  a  positive  reaction  appears  in  twenty- 
four  to  thirty-six  hours,  which  consists  in  a  slight  infiltration  of  skin 
surrounded  by  a  red  areola,  1  to  2  cm.  in  diameter.  A  negative  reaction 
indicates  that  sufficient  natural  antitoxin  is  present  to  protect  the  indi- 
vidual against  diphtheria,  although  he  'may  nevertheless  harbor  the 


578  PATHOGENIC   MICROORGANISMS 

bacilli  as  a  carrier.  It  has  been  found  unnecessary  to  give  prophylactic 
injections  of  antitoxin  to  individuals  with  a  negative  Schick  reaction. 
Park  and  Zingher38  have  found  that  negative  reactions  were  obtained 
in  93  per  cent  of  the  new-born,  and  became  less  frequent  up  to  the  second 
or  fifth  year,  when  37  per  cent  were  negative.  In  older  children  nega- 
tive reactions  were  more  frequently  met  with,  and  about  90  per  cent  of 
adults  were  negative.  A  pseudoreaction  which  appears  earlier  than 
the  true  reaction  and  which  disappears  in  twenty-four  to  forty-eight 
hours  is  occasionally  seen  in  individuals  who  have  natural  antitoxin. 
This  is  due  to  sensitiveness  to  some  of  the  proteins  used  in  the  injection, 
and  may  be  reproduced  in  the  same  individual  by  the  injection  of  autol- 
ysate  of  diphtheria  bacilli  or  sometimes  by  the  injection  of  broth  media. 
The  most  satisfactory  method  for  detection  of  the  pseudoreaction  is  to 
make  a  control  injection  of  Vso  M.L.D.  of  a  toxin  which  has  been 
heated  at  80°  C.  for  five  minutes.  This  heating  destroys  the  toxin,  but 
leaves  uninjured  the  substances  which  produce  the  pseudoreaction. 

The  Schick  reaction  has  been  extensively  used  in  the  last  six  years, 
especially  on  large  bodies  of  troops  and  on  school  children,  and  has  been 
found  eminently  satisfactory,  though,  of  course,  as  with  all  other  biolog- 
ical reactions,  exceptions  are  noted  and  difficulties  encountered.  The 
New  York  Department  of  Health  and  other  health  departments  are 
now  putting  out  materials  for  the  Schick  reaction  in  packets  so  arranged 
that  the  necessary  toxin  is  inclosed  in  capillary  tubes  and  can  be  blown 
out  into  a  mixed  amount  of  salt  solution  with  a  rubber  bulb.  A  part 
of  this  mixture  can  then  be  heated  to  80°  and  an  injection  with  a  control 
done  on  the  upper  arm  of  subjects  as  indicated. 

Notes  on  Specific  Therapy. — Specific  therapy  in  diphtheria  is,  of 
course,  the  main  thing  in  clinical  control.  It  must  be  remembered  here 
as  in  all  other  forms  of  specific  therapy  that  success  depends  as  much 
upon  the  time  at  which  the  diagnosis  is  made  as  it  does  upon  the  manner 
of  treatment.  Therefore,  in  speaking  of  specific  therapy,  it  is  impor- 
tant to  emphasize  the  necessity  for  early  diagnosis.  It  is  in  this  par- 
ticular that  the  responsibility  of  the  physician  is  greatest,  and  every 
severe  sore  throat  in  a  child  should  be  immediately  cultured  and,  though 
not  as  imperative  in  adults,  it  is  not  a  bad  rule  to  culture  all  throats  by 
the  Loeffler  method.  We  have  discovered  a  number  of  cases  in  that  way, 
especially  in  military  sanitation,  which  would  not  have  been  suspected 
clinically.  The  procedure  is  easy,  consumes  no  time  and  requires  so 


38  Park  and  Zingher,  Jour.  A  .M.  A.,  65,  1915,  2216. 


BACILLUS  DIPHTHERIA  579 

little  skill  that  even  in  outlying  districts  the  physician  can  easily  carry 
out  the  steps  himself  with  a  simple  equipment. 

In  order  to  understand  the  practical  principles  of  diphtheria  anti- 
toxin treatment,  it  is  necessary  for  the  physician  to  remember  chiefly 
two  basic  facts.  One  is  the  observation  by  Schick  and  others  that  even 
though  diphtheria  toxin  does  not  enter  so  rapidly  into  combination 
with  the  tissues  of  the  nervous  system,  as  does  tetanus  toxin,  it,  never- 
theless, is  bound  to  some  extent  and  that  the  antitoxin  probably  does 
not  reach  poison  that  is  already  combined  with  tissue  elements.  It  is 
probable  that  the  injury  once  done  is  irretrievable,  at  least  to  a  great 
extent,  and  that  the  antitoxin  is  chiefly  effective  against  the  circulating 
poison  before  such  cellular  attachment  has  been  established.  Experi- 
ments of  Park  and  his  co-workers  have  shown  that  if  rabbits  are  given 
10  lethal  doses  of  diphtheria  toxin,  they  can  be  saved  by  relatively 
small  doses  of  antitoxin  if  this  is  given  just  before  or  with  the  poison. 
As  the  time  between  injection  of  the  poison  and  the  injection  of  the 
antitoxin  grows  longer,  rapidly  increasing  doses  of  antitoxin  are  neces- 
sary and  if  an  hour  or  more  has  elapsed,  no  amount  of  antitoxin  will  save. 
The  second  basic  point  is  the  one  brought  out  by  the  measurements  of 
Henderson  Smith  and  others  which  show  that  antitoxin  subcutaneously 
injected,  is  but  slowly  absorbed  and  reaches  its  maximum  concentration 
in  the  blood  not  much  before  seventy-three  hours  after  injection. 

The  deductions  to  be  made  from  these  considerations  are,  first,  that 
early  diagnosis  must  be  made,  that  it  is  essential  to  get  the  antitoxin 
in  as  early  as  possible,  and  that  when  the  injection  is  made,  it  is  better 
to  give  a  sufficient  amount  at  the  first  dose  than  to  dribble  it  along  in 
insufficient  amounts  with  intervals  of  many  hours  between  doses. 
These  observations  impose  upon  the  physician  great  responsibilities  of 
judgment,  since  in  cases  seen  late  in  the  disease,  with  very  severe  symp- 
toms of  intoxication,  it  may  be  necessary  to  resort  to  intramuscular  or 
intravenous  injections  of  the  antitoxin. 

The  dosage  of  antitoxin  must  vary  according  to  severity  of  the  case, 
the  stage  at  which  it  is  seen,  and  the  age  of  the  patient.  In  severe  cases, 
10,000  to  20,000  units  should  be  injected. 

If  intravenous  injection  is  resorted  to,  precautions  against  the 
occurrence  of  anaphylaxis  must  of  course  be  taken,  but  in  view  of  the 
relatively  slight  danger  of  death  from  horse  serum  injections  in  man,  the 
risk  of  anaphylaxis  in  cases  in  which  intravenous  test  is  actually  indi- 
cated, is  probably  much  less  than  the  risk  of  delaying  the  introduction 
of  antitoxin  into  the  blood.  If  skin  tests  can  be  done  beforehand, 


580  PATHOGENIC   MICROORGANISMS 

they  should  be  done.  They  take  very  little  time  and  give  the  physician 
a  signal  of  possible  danger.  When  such  intravenous  injections  are 
actually  made  in  sensitive  subjects,  it  seems  advisable  to  dilute  the 
serum  50  per  cent  with  sterile  salt  solution,  in  order  to  render  slow 
injection  easier  so  that  the  injection  of  each  cubic  centimeter  may 
occupy  at  least  one  minute. 

Prophylactic  Immunization  in  Diphtheria. — There  are  two  chief 
methods  of  prophylaxis  in  diphtheria  the  first  and  older  consists  in  inject- 
ing 500  to  1000  units  of  antitoxin  subcutaneously.  This  is  carried  out 
on  contacts  with  positive  Schick  reactions  whenever  speed  of  immuni- 
zation is  desired.  It  is  simple  and  the  principles  underlying  it  are 
obvious,  but  the  immunization  is  short-lived  and  constitutes  a  sensiti- 
zation  of  the  subject  with  horse  serum  which  naturally  is  undesirable 
under  modern  conditions. 

There  is  another  method  of  prophylactic  immunization  which  is 
rapidly  growing  in  importance,  and  in  this  country  is  being  at  the  present 
time  very  actively  worked  upon  by  Park  and  his  collaborators  at  the 
New  York  Department  of  Health.  This  method  consists  in  the  active 
immunization  of  individuals  with  overneutralized  mixtures  of  toxin 
and  antitoxin.  The  possibility  of  developing  this  method  is  really 
suggested  in  the  original  procedure  adopted  by  the  New  York  Depart- 
ment of  Health  for  the  production  of  diphtheria  antitoxin  in  horses, 
namely,  the  partial  neutralization  of  the  first  toxin  injections  by  anti- 
toxin. It  was  suggested  some  years  ago  by  Theobald  Smith  that  some 
such  method  might  be  applicable  for  prophylactic  immunization  in 
human  beings,  and  subsequently  the  method  was  developed  by  the  work 
of  Behring.39  Behring  made  his  first  studies  on  animals,  showing  that 
immunization  in  monkeys,  guinea  pigs,  asses,  etc.,  could  be  accom- 
plished by  such  mixtures.  His  own  first  mixtures  were  so  balanced 
that  the  toxic  action  on  guinea  pigs  was  practically  nil.  Subsequently 
Schreiber40  and  others  studied  the  antitoxin  production  in  human 
beings.  It  was  found  that  the  results  of  immunization  were  noticeable 
in  about  two  to  three  weeks,  and  that  2  to  3  injections  of  properly  bal- 
anced mixtures  would  suffice  to  give  a  sufficient  degree  of  immunity  to 
protect  against  ordinary  spontaneous  infection.  Park  and  Zingher41 
originally  recommended  three  injections  at  intervals  of  six  to  seven  days, 

39  Behring,  Deut.  med.  Woch.,  39,  1913. 

40  Kclireiber. 

41  Park  and  Zingher,  Jour.  A.  M.  A.,  5,  1915,  2216;  Park  and  Zingher,  Jour, 
Amer.  Pub,  Health,  6,  1916,  431,  4 


BACILLUS  DIPHTHERIA  581 

consisting  of  1  c.c.  each  of  a  toxin-antitoxin  mixture  so  balanced  that 
there  were  about  4L+  doses  per  cubic  centimeter,  and  that  the  mix- 
ture contained  about  85  per  cent  of  an  L+  dose  per  unit  of  antitoxin. 
They  added  to  these  doses  a  billion  killed  diphtheria  bacilli  in  order  to 
produce  a  certain  degree  of  anti-bacterial  immunity.  With  such  treat- 
ment Schick  reactions  could  be  rendered  negative  within  a  period  of  a 
few  months.  A  few  individuals  did  not  seem  to  respond  by  the  devel- 
opment of  antitoxin  and  remained  Schick  positive. 

Recently,  the  method  has  been  much  perfected  and  it  is  being  inten- 
sively studied  by  Park  and  his  force  of  workers  at  the  present  time.  The 
mixtures  as  recommended  at  the  present  writing  are  such  that  each 
human  dose  of  1  c.c.  contains  at  least  3L+  doses  of  toxin,  so  neu- 
tralized with  antitoxin  that  5  human  doses  (5  c.c.)  injected  into  a 
guinea  pig  of  300  grams  permits  survival  for  at  least  fourteen  or  fifteen 
days,  but  causes  paralysis  and  death  thereafter,  while  1  e.c.  of  the 
actual  human  dose  produced  no  ill  effects.  From  Drs.  Krumwiede  and 
Banzhof,  we  have  the  details  of  the  N.  Y.  Health  Department  require- 
ments which  have  been  accepted  by  the  United  States  Public  Health 
Department  as  standards  at  the  present  moment.  It  must  not  be 
forgotten,  however,  that  by  the  time  this  book  appears  slight  changes 
may  have  been  made  since  we  are  awaiting  the  publication  of  recent 
studies  from  the  Department  of  Health. 

We  are  indebted  to  the  Director  of  the  Hygienic  Laboratory  of  the 
United  States  Public  Health  Service  in  Washington,  Dr.  George  W. 
McCoy,  for  the  following  suggestions  issued  by  the  Public  Health 
Service.  We  include  these  matters  because  accidents  have  occurred 
of  recent  years  by  mistakes  in  the  preparation  of  the  mixtures  and  even 
deaths  have  been  recorded.  We  take  from  the  memorandum  sent  us 
by  Dr.  McCoy,  the  following: 

The  toxin  and  antitoxin  used  should  be  at  least  three  months  old 
from  the  date  of  planting  and  date  of  bleeding,  respectively.  The  L-h 
doses  have  been  found  with  reference  to  the  United  States  standard 
antitoxin.  The  unitage  of  the  antitoxin  to  be  used  should  be  deter- 
mined against  this  toxin  so  that  one  unit  of  the  antitoxin  to  be  used, 
when  mixed  with  the  L-f  dose  of  this  toxin  should  permit  survival  for 
ninety-six  hours  of  25  per  cent  of  the  guinea  pigs  injected.  When  a 
tentative  mixture  of  the  product  has  been  made,  no  further  toxin  shall 
be  added.  Changes  made  should  consist  not  in  the  addition  of  toxin, 
but  in  the  reduction  of  toxicity  by  the  addition  of  antitoxin,  if  necessary. 
Approximately  one  unit  of  antitoxin  is,  therefore,  added  to  start  with, 


582  PATHOGENIC   MICROORGANISMS 

for  each  L+  dose  in  the  bulk  toxin,  and  with  each  addition  of  antitoxin 
to  the  mixture  in  bulk,  it  shall  be  immediately  thoroughly  shaken  for 
twenty  minutes.  After  each  addition  to  the  mixture,  or  after  the 
original  mixing,  the  preparation  should  stand  at  least  twenty-four  hours 
in  order  to  allow  combination  to  take  place  before  tests  on  guinea  pigs 
are  made.  Six  guinea  pigs  are  then  injected  receiving,  respectively, 
0.1,  0.2,  0.5,  1.0,  2.0,  and  5.0  c.c.  If  all  six  guinea  pigs  die  in  seventy- 
two  hours,  add  0.1  the  former  antitoxin  used,  mix  and  retest.  Continue 
this  until  the  5  c.c.  pig  survives  ninety-six  hours  and  at  this  point  the 
mixture  is  probably  ready  for  filtering.  After  filtering,  the  mixture  is 
preserved  until  at  least  one-half  of  the  5  c.c.  guinea  pigs  on  each  bulk 
bottle  survive  ten  days,  but  show  definite  diphtheritic  paralysis  there- 
after. The  human  dose  is  1.  c.c.,  and  three  doses  a  week  apart  are 
usually  given. 

Bacteria  Similar  to  Bacillus  Diphtherise. — BACILLUS  HOFFMANNI 
(Pseudodiphtheria  bacillus). — Hoffmann- Wellenhoff,42  in  1888,  and,  at 
almost  the  same  time,  Loeffler,43  described  bacilli  which  they  had  culti- 
vated from  the  throats  of  normal  persons  and  in  several  instances  from 
those  of  diphtheritic  persons,  which  were  in  many  respects  similar  to 
true  B.  diphtheria,  but  differed  from  this  chiefly  in  being  non-pathogenic 
for  guinea-pigs.  These  organisms  were  at  first  regarded  by  some 
observers  as  merely  attenuated  diphtheria  bacilli.  More  recent  inves- 
tigations, however,  prove  them  to  be  unquestionably  a  separate  species, 
easily  different iable  by  proper  methods.  They  differ  from  B.  diph- 
therise  in  so  many  important  features,  moreover,  that  the  term  "pseu- 
dodiphtheria  bacillus  "  is  hardly  an  appropriate  one  for  them. 

Morphology. — Bacillus  Hoffmanni  is  shorter  and  thicker  than  Bacillus 
diphtheria.  It  is  usually  straight  and  slightly  clubbed  at  one  end, 
rarely  at  both.  Stained  with  Loeffler's  blue  it  occasionally  shows 
unstained  transverse  bands;  unlike  B.  diphtheriae,  however,  these  bands 
hardly  ever  exceed  one  or  two  in  number  at  most.  In  many  cultures 
the  single  transverse  band  gives  the  bacillus  a  diplococcoid  appearance. 

Staining. — Stained  by  Neisser's  or  Roux's  method,  no  polar  bodies 
can  be  demonstrated.  The  bacillus  forms  no  spores,  is  non-motile, 
and  possesses  no  flagella. 

Cultivation. — On  the  usual  culture  media  B.  Hoffmanni  grows  more 
luxuriantly  than  B.  diphtheria,  developing  even  in  first  isolations  from 


*  Hoffmann-Wellenhoff,  Wien.  med.  Woch<,  iii,  1888. 
"  Loeffler,  Cent.  f.  Bakt.,  ii,  1887. 


BACILLUS   DIPHTHERIA  583 

the  human  body  upon  the  simple  meat-extract  media.  On  agar  plates 
its  colonies  are  larger,  less  transparent,  and  whiter  than  are  those  of 
true  diphtheria  bacilli.  In  fluid  media  there  is  even  clouding  and  less 
tendency  to  the  formation  of  a  pellicle  than  with  B.  diphtherise.  A 
positive  means  of  distinction  between  the  two  is  given  by  the  inability 
of  B.  Hoffmanni  to  form  acid  upon  various  sugar  media.  The  differ- 
entiation on  a  basis  of  acid  formation  was  first  attempted  by  Cobbett44 
and  has  been  recently  worked  out  systematically  by  Knapp,45  and  con- 


FIG.  61. — BACILLUS  HOFFMANNI.  FIG.  62. — COLONIES  OF  BACILLI 

HOFFMANNI  ON  AGAR. 


firmed  by  various  observers.46  The  results  of  this  work,  carried  out 
with  the  serum-water  media  of  Hiss,  to  which  various  sugars  were  added, 
show  that  B.  Hoffmanni  forms  acid  upon  none  of  the  sugars  used,  while 
B.  diphtheria  acidifies  and  coagulates  media  containing  monosaccharids 
and  several  of  the  more  complex  sugars,  as  given  in  the  diagram  in  the 
section  following,  dealing  with  B.  xerosis. 

Differentiation  can  finally  be  made  on  the  basis  of  animal  patho- 
genicity,  B.  Hoffmanni  being  entirely  innocuous  to  the  ordinary  labora- 


44  Cobbett,  Cent,  f .  Bakt.,  1898. 

^Kuapp,  Jour.  Med.  Kes.,  vii,  1904. 

46  Graham  Smith,  Jour,  of  Hyg.,  vi,  1906;  Zinsser,  Jour,  Med,  Res.,  xvii,  1907. 


584  PATHOGENIC  MICROORGANISMS 

tory  animals.     B.  Hoffmann!  forms  no  toxins,  and  animals  immunized 
with  it  do  not  possess  increased  resistance  to  B.  diphtheriae. 

BACILLUS  XEROSIS. — In  1884,  Kutschert  and  Neisser47  described  a 
bacillus,  isolated  from  the  eyes  of  patients  suffering  from  a  form  of 
chronic  conjunctivitis  known  as  xerosis.  This  bacillus,  which,  morpho- 
logically, is  almost  identical  with  B.  diphtheriae,  they  believed  to  be  the 
etiological  factor  of  the  disease.  The  frequency  with  which  it  has  been 
isolated  from  normal  eyes,  precludes  this  etiological  relationship,  and 
it  may  safely  be  regarded  as  a  harmless  parasite  which  may  indeed  be 
more  abundant  in  the  slightly  inflamed  than  in  the  normal  conjunctiva. 
Morphology. — B.  xerosis  resembles  B.  diphtherias  closely.  It  is 
occasionally  shorter  than  this,  but  on  the  whole  no  absolute  morpho- 
logical differentiation  between  the 
two  is  possible.  It  forms  no  spores 
and  is  non-motile.  Polar  bodies  may 
occasionally  be  seen. 

Cultivation'— On  Loeffler's  blood 
serum,  on  agar,  glycerin  agar,  and  in 
broth,  its  growth  is  very  similar  to 
that  of  B-  diphtheriae,  but  more  del- 
icate  throughout.  It  cannot  easily 
be  cultivated  upon  the  simple  meat- 
FIG.  63.— BACILLUS  XEROSIS.  extract  media,  nor  will  it  grow  on 

gelatin   at    room    temperature.     Its 

colonies  on  glycerin  or  glucose  agar  are  microscopically  identical  with 
those  of  B.  diphtheriae. 

Differentiation. — It  differs  from  B.  diphtheriae  distinctly  in  its  acidi- 
fying action  on  sugar  media.  These  relations  were  first  worked  out  by 
Knapp  for  various  sugars  and  the  alcohol  mannit,  and  have  been  exten- 
sively confirmed  by  others.  See  table  on  page  585. 

A  reference  to  the  table  shows  that  differentiation  may  be  made 
by    the  use  of  two  sugars — saccharose  and  dextrin.     B.   diphtheriae 
forms  acid  from  dextrin,  not  from  saccharose;   B.  xerosis  from  saccha- 
rose, not  from  dextrin;  B.  Hoffmanni  does  not  form  acid  from  either. 
B.  xerosis  is  non-pathogenic  to  animals  and  forms  no  toxin. 
The  Diphtheroid  Bacilli. — In  addition  to  the  bacteria  mentioned 
above,  there  is  a  large  group  of  microorganisms  spoken  of  as  the  diph- 
iheroid  bacilli,  largely  because  of  their  morphological  resemblance  to 
the  diphtheria  bacillus.     For  this  group,  Lehman  and  Neumann  have 

"Kutschert  und  Neisser,  Deut.  med.  Woch.,  xxiv,  1884. 


BACILLUS  MPHTHERLE 


585 


suggested  the  term  corynebacterium.  The  characteristics  of  this  group 
are  a  morphological  similarity  to  the  diphtheria  bacillus,  that  they  are 
Gram-positive,  non-motile,  often  show  metachromatic  granules  and 
have  no  spores.  It  is  not,  at  the  present  writing,  possible  to  formulate 
a  classification  of  these  organisms.  They  are  apparently  very  numer- 
ous and  have  been  isolated  from  a  great  many  different  sources,  both 


Hiss  serum-water  media  plus  1% 

B.  Diphtheria; 

B.  Xerosis 

B.  Hoffmanni 

Dextrose 

+ 

4. 

Saccharose                   

+ 

Dextrin                                                  .  . 

+ 

in  connection  with  the  human  body  and  in  nature.  Recently  Bunting 
and  Yates  have  claimed  that  an  organism  of  this  group  has  etiological 
connection  with  Hodgkin's  disease.  Studies  by  many  other  workers, 
notably  by  Bloomfield  and  Fox,-  and  studies  going  on  in1  our  own  labora- 
tory show  that  organisms  very  similar  to  these  strains  can  be  isolated 
from  the  skin,  from  the  lymph  nodes  of  healthy  and  diseased  people, 
from  ascitic  fluid  in  varying  conditions,  and  from  supposedly  sterile 
tissues.  They  are  frequently  present  in  the  nasal  mucus  and  in  the 
throat,  and  are  so  ubiquitous  that  any  association  of  them  with  specific 
disease  must  be  very  conservatively  approached.  According  to  the 
investigations  of  many  men  who  have  studied  the  flora  of  the  naso- 
pharynx, it  seems  that  organisms  belonging  to  the  general  group  of 
diphtheroid  bacilli  are  the  most  common  saprophytes  habitually  present 
in  this  part  of  the  normal  human  body. 

Very  similar  to  this  group  are  the  bacilli  of  pseudo-tuberculosis  ovis, 
isolated  from  necrotic  lesions  in  the  kidneys  of  sheep  by  Preisz  and 
Nocard. 

It  is  impossible  at  present  to  do  more  than  indicate  that  the  "  diph- 
theroid bacilli"  are  a  large  heterogeneous  group,  held  together  by 
morphological  and  superficial  cultural  similarity  and  largely  consisting 
of  saprophytes  and  probably  harmless  parasites  on  the  human  and 
animal  body. 

Recently,  Mallory  and  Frederic  Parker  have  isolated  diphtheroid 
bacilli  from  middle  ear  lesions  secondary  to  scarlet  fever  which  seem  to 
deserve  more  than  ordinary  attention  because  of  their  apparent  produc- 
tion of  a  powerful  toxic  substance.  These  organisms  are  now  being 
studied  in  our  laboratory  by  Dr.  Parker. 


CHAPTER  XXIX 

THE  TUBERCLE  BACILLUS 

IN  view  of  the  'clinical  manifestations  of  tuberculosis,  it  is  not  sur- 
prising that  the  infectious  nature  of  the  disease  has  been  suspected  for 
many  centuries.  Even  Fracastor  had  remarkably  modern  ideas  con- 
cerning its  transmission.  Inoculation  by  means  of  tuberculous  material 
was  first  successfully  accomplished  by  Klencke,  in  1843,  and,  more  elabo- 
rately, by  Villemin,1  in  1865.  It  was  not  until  1882,  however,  that 
Koch2  succeeded  in  isolating  and  cultivating  the  tubercle  bacillus. 
Baumgarten  3  had  previously  seen  the  bacillus  in  tissue  sections,  but  his 
researches  were 'limited  to  purely  morphological  observations.  Koch, 
in  addition  to  demonstrating  the  bacillus  in  tuberculous  tissues  from 
various  sources,  produced  characteristic  lesions  in  guinea-pigs  and  other 
animals  by  infecting  them  with  pure  cultures,  and  established  beyond 
doubt  the  etiological  relationship  of  the  bacillus  to  the  disease. 

Morphology. — Tubercle  bacilli  appear  as  slender  rods,  2  to  4  micra 
in  length,  0.2  to  0.5  micra  in  width.  Their  ends  are  usually  rounded. 
The  rods  may  be  straight  or  slightly  curved;  their  diameters  may  be 
uniform  throughout;  more  often,  however,  they  appear  beaded  and 
irregularly  stained.  The  beaded  appearance  is  due  to  different  causes. 
Unstained  spaces  may  occur  along  the  body  of  the  bacillus,  especially 
in  old  cultures.  These  are  generally  regarded  as  vacuoles.  The  bodies 
of  the  bacilli,  on  the  other  hand,  may  bulge  slightly  here  and  there,  often 
in  three  or  four  places,  showing  oval  or  rounded  knobs  which  stain  with 
great  depth  and  are  very  resistant  to  decolorization.  These  thiqkenings 
were  formerly  regarded  as  spores,  but  in  view  of  the  fact  that  the  bacilli 
are  not  more  resistant  against  heat  and  disinfectants  than  other  vegeta- 
tive forms,  this  interpretation  is  probably  incorrect.  The  bacilli  are 
said  to  possess  a  cell  membrane  which  confers  upon  them  their  resistance 


1  Villemin,  Gaz.  hebdom.,   1865. 

2  Koch,  Berl.  klin.  Woch.,  1882  ;  Mitt.  a.  d.  kais.  Gesundheitsamt,  1884. 

3  Baumgarten,  Virchow's  Arch.,  Ixxxii. 

586 


THE  TUBERCLE  BACILLUS  587 

against  drying  and  entrance  of  stains.  This  membrane  gives  a  cellulose 
reaction  and  is  believed  to  contain  most  of  the  waxy  substances  which 
can  be  extracted  from  the  cultures. 

Various  observers,  notably  Nocard  and  Roux,4  Mafucci,5  and  Klein,6 
have  demonstrated  branched  forms  of  the  tubercle  bacillus.  These 
observations,  variously  extended  and  confirmed,  make  it  probable  that 
Bacillus  tuberculosis  is  not  a  member  of  the  family  of  schizomycetes, 
but  belongs  rather  to  the  higher  bacteria,  closely  related  to  the  actino- 
myces. 

Staining. — Tubercle  bacilli  do  not  stain  easily  with  the  ordinary 
anilin  dyes;  to  these  they  are  made  permeable  only  by  long  exposure 
or  by  heating  of  the  staining  solution.  Once  stained,  however,  the  dye 
is  tenaciously  retained  in  spite  of  treatment  with  alcohol  and  strong 
acids.  For  this  reason,  this  bacillus,  together  with  some  other  bacteria 
to  be  mentioned  later,  is  spoken  of  as  "acid-fast."  The  acid-fast  nature 
of  the  bacillus  seems  to  depend  upon  the  fatty  substances  contained  in 
it,7  and  has  furnished  the  basis  for  differential  staining  methods.  All 
the  staining  methods  devised  for  the  recognition  of  the  tubercle  bacillus 
thus  depend  upon  the  use  of  an  intensely  penetrating  staining  solution, 
followed  by  vigorous  decolorization  which  deprives  all  but  the  acid-fast 
group  of  their  color.  Counterstains  of  any  of  the  weaker  dyes  may 
then  be  used  to  stain  the  decolorized  elements.  One  of  the  first  of  the 
staining  solutions  to  be  of  practical  use  was  the  anilin-water-gentian- 
violet  solution  of  Ehrlich  8  (11  c.c.  saturated  alcoholic  gentian-violet 
to  89  c.c.  5  per  cent  anilin  water).  This  dye,  although  of  sufficient 
penetrating  power,  has  the  disadvantage  of  deteriorating  rapidly  and 
has  in  practice  been  almost  entirely  displaced  by  Ziehl's  9  carbol-fuchsin 
solution.  (Fuchsin  1  gm.  in  10  c.c.  alcohol  absolute,  added  to  90  c.c. 
5  per  cent  carbolic.)  This  staining  solution  is  the  one  now  in  general 
use  and  is  employed  as  follows:  Thin  smears,  on  slides  or  cover-slips, 
are  covered  with  the  dye  and  gently  heated.  In  the  case  of  cover-glasses, 
these  may  be  floated,  face  downward,  in  staining  dishes  filled  with  the 
dye.  The  dye  is  allowed  to  act  for  about  three  minutes,  steaming  but  not 
allowed  to  boil.  At  the  end  of  this  time  the  preparation  is  washed 

4  Nocard  et  Roux,  Aim.  <le  1'inst.  Pasteur,  1887. 

*  Mafucci,  Zeit.  f.  Hyg.,  ii. 
'Klein.  (Vnt.   f.    Uakt.,   1890. 

7  />'/r;/.v/or/,,    1'WI.   (1.   M<M!.,    l,XS(j;    /JY///.    Dciil.    nicd.    \Vorli.,    1SP1. 

*  Khi-lifli,   Di-iil.  tuc.l.   Woch.,   18SL> ;    ll'cif/crl,  I>out.  uio.l.   \Vnch.,   .Iss.l. 

9  Xichl,  Deut,  nied.  Woch.,  1883;  Ncelscn,  "Lehrb.  <1.  allff.  Path.,"  1894, 


588  PATHOGENIC   MICROORGANISMS 

either  with  5  per  cent  nitric  acid,  5  to  20  per  cent  sulphuric  acid,  or  1 
per  cent  hydrochloric  acid,  until  most  of  the  red  color  has  disappeared 
(a  few  seconds),  and  the  preparation  appears  pale  pink.  This  results  in 
decolorization  of  all  microorganisms  with  the  exception  of  members  of 
the  acid-fast  group.  Thorough  washing  in  80  to  95  per  cent  alcohol 
is  now  employed  to  complete  the  decolorization.  The  preparation  is 
then  rinsed  in  water  and  counterstained  with  1  per  cent  aqueous  methy- 
lene-blue. 

Tubercle-bacillus  staining  has  been  further  simplified  by  Gabbett,10 
who  combines  decolorization  and  counterstaining.  In  this  method 
preparations  are  stained  with  Ziehl's  carbol-fuchsin  as  in  the  preceding; 
they  are  then  rinsed  in  water  and  covered  with  a  solution  containing 
methylene-blue  1  gram,  concentrated  sulphuric  acid  25  grams,  and 
distilled  water  100  c.c.  This  is  allowed  to  act  for  from  two  to  four  min- 
utes, at  the  end  of  which  time  all  elements  in  the  preparation  except 
the  acid-fast  bacilli  will  be  decolorized  and  counterstained. 

Another  excellent  stain  for  tubercle  bacilli,  which  has  the  advantage 
of  greater  clearness  of  contrast  over  the  carbol-fuchsin  stain  is  that  of 
Hermann  in  which  Crystal  Violet  is  used.  It  is  described  in  the  section 
on  staining. 

Tubercle  bacilli  in  very  young  culture  are  often  not  acid-fast  and  it 
is  not  always  possible  to  demonstrate  acid-fast  bacilli  in  pus  from  cold 
abscesses  in  sputum,  in  serous  exudates,  and  in  granulomatous  lesions 
of  the  lymph  nodes  which  can  be  shown  by  animal  inoculation  to  be 
tuberculous.  Much n  demonstrated  in  such  material  Gram-positive 
granules  which  lay  singly,  in  short  chains  or  in  irregular  clumps,  and 
which  he  believed  to  be  non-acid-fast  tubercle  bacilli.  He  found  similar 
granules  in  cultures  of  tubercle  bacilli  which  showed  on  further  incuba- 
tion numerous  acid-fast  bacillary  forms.  His  work  has  been  repeatedly 
confirmed,  and  there  seems  little  doubt  but  that  these  granules  are  really 
tubercle  bacilli.  Their  demonstration  is  not,  however,  of  great  diag- 
nostic value,  as  other  bacilli  form  granules  of  the  same  appearance. 
Small  rods  and  splinters  are  also  found  which  stain  by  Gram's  method, 
but  not  by  carbol-fuchsin.12 

To  find  "Much's  granules,"  smears  or  sections  are  steamed  in  a 
solution  of  methyl  violet  B.N.  (10  c.c.  of  saturated  alcoholic  solution 
of  the  dye  in  100  c.o.  of  distilled  water  containing  2  per  cent  phenol). 

10  Gabbett,  Lancet,  1887. 

11  Much,  Ben.  klin.  Woch.,  1908,  xlv,  700. 

12  Liebermeister,  Deutsche  med.  Woch.,  1909,  xxxv,  1324, 


THE  TUBERCLE  BACILLUS  589 

They  are  then  treated  with  Gram's  iodine  solution  one  to  five  minutes; 
5  per  cent  nitric  acid  one  minute;  3  per  cent  hydrochloric  acid  ten  sec- 
onds; absolute  alcohol  and  acetone  equal  parts,  until  decolorized.  The 
granules  may  be  stained  by  other  modifications  of  Gram's  method. 
Weiss  13  has  devised  a  combination  stain.  One  part  of  Much's  methyl 
violet  is  mixed  with  three  parts  of  ZiehFs  carbol-fuchsin  and  filtered; 
slides  are  stained  for  twenty-four  to  forty-eight  hours  in  the  mixture. 
They  are  then  decolorized  as  in  Much's  method  and  counterstained  with 
Bismarck  brown  or  safranin  1  per  cent.  Both  acid-fast  and  Gram- 
positive  forms  are  stained  by  this  method  and  in  the  red  may  be  seen 
blue-black  granules. 

While  the  acid-fast  group  of  bacteria  is  composed  of  a  number  of 
organisms  to  be  mentioned  later,  a  few  only  of  these  offer  difficulties  of 
differentiation  from  the  tubercle  bacillus.  Those  to  be  considered 
practically  are  the  bacillus  of  leprosy  and  that  of  smegma.  The  latter 
bacillus,  because  of  its  distribution,  is  not  infrequently  found  to  con- 
taminate feces,  urine,  or  even  sputum,  and  it  is  sometimes  desirable  to 
apply  to  suspected  specimens  one  or  the  other  of  the  stains  devised 
for  the  differentiation  of  the  smegma  bacillus  from  Bacillus  tuber- 
culosis. The  one  most  frequently  employed  is  that  of  Pappenheim.14 
The  preparations  are  stained  in  hot  carbol-fuchsin  as  before;  the 
carbol-fuchsin  is  then  poured  off  without  washing  and  the  preparation 
immersed  in  solution  made  by  saturating  a  1  per  cent  alcoholic  solu- 
tion of  rosolic  acid  with  methylene-blue  and  adding  20  per  cent  of 
glycerin.  In  such  preparations  tubercle  bacilli  remain  red,  smegma 
bacilli  appear  blue. 

Stained  by  Gram,  tubercle  bacilli  retain  the  gentian- violet. 

When  tubercle  bacilli  are  very  sparsely  present  in  sputum  and  other 
material  it  may  be  impossible  to  find  them  by  direct  examination, 
and  often  the  only  method  of  finding  them  will  be  animal  inoculation. 
However,  a  number  of  methods  have  been  devised  by  which  the  bacilli 
may  be  concentrated  in  such  a  way  that  they  may  be  found  even  when 
a  few  only  are  present.  One  of  these  is  to  add  peroxide  of  hydrogen  to 
the  sputum.  By  this  the  mucus  is  dissolved  out  and  the  solid  particles 
sstth  or  may  be  centrifugalized.  A  method  very  commonly  employed 
to-day  is  that  which  depends  on  the  use  of  "antiformin."  This  is  a 
preparation  used  extensively  for  the  cleansing  of  vats  in  breweries.  It 


13  Weiss,  Bcrl.  klin.  Woch.,  1909,  xlvi,   1797. 

14  Pappenheim,  Berl.  klin.  Woch.,  1898. 


590  PATHOGENIC   MICROORGANISMS 

is  described  by  Rosenau  15  as  consisting  of  equal  parts  of  liquor  sodse 
chlorinatse  and  a  15  per  cent  solution  of  caustic  soda.  The  formula  for 
liquor  sodse  chlorinatse  he  gives  as : 

Sodium  carbonate 000 

Chlorinated  lime 400 

Distilled  water   4,000 

If  sputum  is  poured  into  a  10  to  15  per  cent  solution  of  antiformin 
and  allowed  to  stand  for  several  hours,  most  of  the  other  elements  of 
the  sputum,  cells,  and  bacteria,  will  dissolve  out,  and  acid-fast  bacilli 
be  left  in  the  residue.  'Strangely  enough  they  are  not  killed  by  this 
process  and  if  sufficiently  washed  may  be  cultivated  or  can  produce 
lesions  in  guinea-pigs. 

Isolation  and  Cultivation. — Tubercle  bacilli  are  not  easily  cul- 
tivated. Their  slowness  of  growth  precludes  isolation  by  plating.  The 
first  isolations  by  Koch  16  were  made  upon  coagulated  blood  serum  from 
tuberculous  tissue. 

Isolation  from  tuberculous  material  may  be  aided  by  inoculation  into 
guinea-pigs.  These  animals  will  withstand  the  acute  infection  produced 
by  the  contaminating  organisms  and  succumb  later  (four  to  six  weeks) 
to  tuberculosis.  The  bacilli  may  then  be  obtained  by  cultivations  from 
lymph  nodes  or  other  foci  which  contain  only  tubercle  bacilli.  When 
isolation  from  sputum  is  attempted  whether  directly  or  by  means  of 
animal  inoculation,  the  sputum  may  be  rendered  comparatively  free 
from  contaminating  bacteria  by  washing.  The  sputum  is  rinsed  in 
running  water  to  free  it  from  pharyngeal  mucus.  It  is  then  washed 
in  eight  or  ten  changes  of  sterile  water.  The  material  selected  is  taken 
from  the  center  of  the  washed  mass,  if  possible  from  the  flakes  of  caseous 
material  visible  in  such  sputum. 

For  the  isolation  of  tubercle  bacilli  from  sputum  and  other  materials  in 
which  contaminating  bacteria  of  other  species  are  present,  Petroff17  has 
devised  an  excellent  method  which  has  been  tried  out  and  used  with  success 
in  our  laboratory.  The  principles  on  which  PetrofPs  method  rests  are,  first 
of  all,  the  bactericidal  power  of  3  per  cent  sodium  hydroxid  on  non-acid-fast 
bacteria,  and  the  selective  action  of  dyes  like  gentian  violet  on  bacterial 
growth,  as  first  practically  utilized  by  Churchman. 

15  Rosenau,  ' '  Preventive  Medicine  and  Hyigene, ' '  D.  Appleton,  X.  Y.,   1913 ; 
Uhlenhuth,  Berl.  klin.  Woch.,  Xo.  29,  1908. 
18  Koch,  loc  cit. 
17  Petroff,  Johns  Hopkins  Hosp.  Bull.,  vol.  xxvi,  Xo.  294,  August,  1915,  p.  276. 


THE   TUBERCLE   BACILLUS  591 

The  medium  used  by  Petroff  is  made  as  follows: 

I.  Meat  Juice.     Five  hundred  grams  of  beef  or  veal  are  infused  in  500 
c.c.  of  a  15  per  cent  solution  of  glycerin  in  water,  in  a  cool  place.     After 
twenty-four  hours  the  meat  is  squeezed  in  a  sterile  press  and  the  infusion 
collected  in  a  sterile  beaker. 

II.  Eggs.  The  shells  of  the  eggs  are  sterilized  by  ten  minute  immersion 
in  70.  per  cent  alcohol.     They  are  broken  into  a  sterile  beaker,  well  mixed 
and  filtered  through  sterile  gauze.     One  part  of  meat  juice  is  added  to  two 
parts  of  egg  by  volume. 

III.  Gentian   Violet.     One  per  cent  alcoholic  solution   of  gentian  violet 
is  added  to  make  a  final  proportion  of  1 :10,000. 

The  three  ingredients  are  well  mixed.  The  medium  is  tubed  and  inspissated 
as  usual. 

Petroff  recommends  for  sputum  the  following  technique:  Equal  parts  of 
sputum  and  3  per  cent  sodium  hydroxid  are  shaken  and  incubated  at  38°  C. 
for  fifteen  to  thirty  minutes,  the  time  depending  on  the  consistency  of  the 
sputum.  The  mixture  is  neutralized  to  litmus  with  hydrochloric  acid  and 
centrifugalized.  The  sediment  is  inoculated  into  the  medium  described  above. 
Pure  cultures  are  obtained  in  a  large  proportion  of  cases. 

PetrofFs  method  has  been  applied  by  him  to  feces,  in  which  the  problem 
is  made  more  difficult  by  the  presence  of  many  spore-formers  which  resist 
sodium  hydroxid.  Feces  is  collected  and  diluted  with  three  volumes  of  water, 
and  then  filtered  through  several  thicknesses  of  gauze.  The  filtrate  is  saturated 
with  sodium  chlorid  and  left  for  half  an  hour.  The  floating  film  of  bacteria 
is  collected  in  a  wide-mouthed  bottle  and  an  equal  volume  of  normal  sodium 
hydroxid  is  added.  This  is  shaken  and  left  in  the  incubator  for  three  hours, 
shaking  every  half  hour.  It  is  then  neutralized  to  litmus  with  normal 
hydrochloric,  centrifugalized,  and  the  sediment  planted. 

Once  isolated,  the  bacilli  are  best  grown  on  glycerin  egg  medium 
which  is  described  in  the  section  on  Media.  On  this  medium  colonies 
of  the  human  bacilli  begin  to  appear  after  six  or  eight  days  as  yellowish 
white  moist  crust-like  flakes. 

On  blood  serum  at  37.5°  C.,  colonies  become  visible  at  the  end  of  eight 
to  fourteen  days.  They  appear  as  small,  dry,  scaly  spots  with  corru- 
gated surfaces.  After  three  or  four  weeks,  these  join,  covering  the  sur- 
face as  a  dry,  whitish,  wrinkled  membrane.  Coagulated  dog  serum  is 
regarded  by  Theobald  Smith  18  as  a  favorable  media  for  the  growth  of 
tubercle  bacilli. 


ls  Tli.  NM////J,  .lour.    Hxp.   Mod.,  iii,  1898. 


592 


PATHOGENIC   MICROORGANISMS 


Slants  of  agar,  to  which  whole  rabbit's  blood  has  been  added  in  quan- 
tities of  from  1  to  2  c.c.  to  each  tube,  make  an  excellent  medium. 

Cultivation  methods  were  simplified  by  the  discovery  by  Roux  and 
Nocard  that  glycerin  facilitates  cultivation.  Upon  glycerin-cigar 
(glycerin  3  to  6  per  cent),  at  37.5°  C.,  colonies  become  visible  at  the  end 
of  from  ten  days  to  two  weeks. 

Glycerin  bouillon  (made  of  beef  or  veal  with  pepton  1  per  cent, 

glycerin  6  per  cent,  slightly  alkaline) 
is  a  favorable  medium.  It  should  be 
filled,  in  shallow  layers,  into  wide- 
mouthed  flasks,  since  oxygen  is  essen- 
tial. Transplants  to  this  medium  should 
be  made  by  carefully  floating  flakes  of 
the  culture  upon  the  surface.  In  this 
medium  the  bacilli  will  spread  out 
upon  the  surface,  at  first  as  a  thin, 
opaque,  floating  membrane.  This 
rapidly  thickens  into  a  white,  wrinkled, 
or  granular  layer,  spreading  over  the 
entire  surface  of  the  fluid  in  from  four 
to  six  weeks.  Later,  portions  of  the 
membrane  sink.  In  old  cultures,  the 
membrane  becomes  yellowish.  These 
cultures  emit  a  peculiar  aromatic 
odor.  Cultures  when  first  grown  on 
solid  media  are  a  little  difficult  to  start 
on  glycerin  broth.  To  accomplish  this 
it  is  best  to  grew  them  for  a  few 
weeks  on  egg  or  glycerin  egg  slants 
containing  considerable  amounts  of 
condensation  water.  At  the  end  of 
this  time  the  growth  will  have  begun 

to  grow  over  the  surface  of  the  condensation  water,  and  from  this 
pellicle  a  bit  can  be  picked  up  with  a  bent  loop,  carefully  removed  with- 
out allowing  it  to  immerse  in  the  fluid  and  this  carefully  floated  on  the 
surface  of  the  glycerin  broth  in  the  flask. 

Glycerin  potato  forms  a  favorable  culture  medium  for  the  bacillus. 
Hesse  19  has  devised  a  medium  containing  a  proprietary  preparation 


FIG.  64. — CULTURE  OF  BACILLUS 
TUBERCULOSIS  IN  FLASK  OF 
GLYCERIN  BOUILLON. 


18  Hesse,  Zeit.  f.  Hyg..  xxxi. 


THE  TUBERCLE  BACILLUS  593 

known  as  "Nahrstoff  Heyden/'  upon  which  tubercle  bacilli  are  said  to 
proliferate  more  rapidly  than  other  bacteria.  His  method  has  yielded 
excellent  results.  It  is  prepared  as  follows : 

' '  Nahrstoff  Heyden "  -° 10  grams 

Sodium  chlorid   5    'll 

Glycerin 30      " 

Ag-ar   10      l ' 

Normal  sodium  solution 5     c.c. 

Aq.  dcst  1,000      < ' 

Biological  Considerations. — The  tubercle  bacillus  is  dependent 
upon  the  access  of  oxygen.  Its  optimum  temperature  is  37.5°  C. 
Temperatures  below  30°  and  above  42°  C.  inhibit  its  growth.  In  fluid 
media,  the  bacilli  are  killed  by  60°  in  fifteen  to  twenty  minutes,  by  80° 
in  five  minutes,  by  90°  in  one  to  two  minutes.  They  will  withstand  dry 
heat  at  100°  C.  for  one  hour.  They  are  resistant  to  cold.  The  com- 
paratively high  powers  of  resistance  of  the  bacillus  are  attributed  to  the 
protective  qualities  of  the  waxy  cell  membrane.21 

The  life  of  cultures,  kept  in  favorable  environment,  is  from  two  to 
eight  months,  varying  to  some  extent  with  the  nature  of  the  culture 
medium.  The  viability  of  the  bacilli  in  sputum  is  of  great  hygienic 
importance.  In  most  sputum  they  may  remain  alive  and  virulent  for  as 
long  as  six  weeks,  in  dried  sputum  for  more  than  two  months.22 

Five  per  cent  carbolic  acid  kills  the  bacilli  in  a  few  minutes.23  Used 
for  sputum  disinfection,  where  the  bacilli  are  protected,  complete  disin- 
fection requires  five  to  six  hours.  Bichloride  of  mercury  is  not  very 
efficient  for  sputum  because  of  the  formation  of  albuminate  of  mercury. 
Direct  sunlight  kills  in  a  few  hours. 

Pathogenicity. — The  tubercle  bacillus  gives  rise  in  men  and  sus- 
ceptible animals  to  specific  inflammations  which  are  so  characteristic 
that  a  diagnosis  of  tuberculosis  may  be  made  by  histological  examina- 
tion, even  though  tubercle  bacilli  themselves  are  not  found.  These 
foci,  known  as  tubercles,  were  first  studied  in  detail  by  Baumgarten,24 
and,  since  then,  have  been  the  object  of  many  pathological  investiga- 

20 "  Nahrstoff  Heyden ';  is  prepared  in  Germany.  It  is  a  white  powder  similar 
to  nutrose. 

21  Tli.  Smith,  Jour.  Exper.  Med.,  1899 ;   Grancher  et  Ledoux-Lebard,  Arch,  de 
med.  exper.,  1892;   Galtier,  Compt.  rend,  de  1'acad.  des  sci.,  1887. 

22  Schell  und  Fischer,  Mitt.  a.  d.  kais.  Gesundheitsamt,  1884. 

23  De  Toma,  Ann.  di  med.,  1886. 

*4  Baumgarten,  Bcrl.  klin.  Woch.,  1901. 


594  PATHOGENIC   MICROORGANISMS 

tions.  The  lesions  are  fundamentally  alike  wherever  they  occur,  though 
in  their  detailed  histological  structure  they  may  vary  somewhat  accorck 
ing  to  the  tissue  in  which  they  appear.  They  begin  as  microscopic 
agglomerations  of  concentrically  arranged  epithelium-like,  or  epitheloid 
cells,  around  which  eventually  lymphocytes  accumulate.  These  micro- 
scopic tubercles  may  gradually  enlarge  individually,  or  they  may  grow 
by  coalescence  with  neighboring  tubercles.  Characteristic  giant  cells, 
with  marginal  centers  of  nuclei,  appear  near  the  centers  of  the  tubercles 
and  in  these  giant  cells  the  tubercle  bacilli  are  usually  found.  As  the 
tubercles  grow  in  size,  the  central  mass  becomes  necrotic.  Fluid  pus 
does  not  form,  and  the  centers  assume  a  grumous  and  friable  condition 
which  is  generally  described  as  caseous  or  cheesy.  Such  tubercles  may 
result  from  the  injection  of  dead  bacilli,  as  well  as  living  ones  as  Prudden 
and  Hodenpyl  have  shown.  The  cheesy  degeneration  may  be  in  part 
due  to  the  toxic  action  of  the  substances  of  the  tubercle  bacilli  and  in 
part  by  pressure  and  lack  of  vascularization.  It  is  astonishing  how 
difficult  it  is  to  find  tubercle  bacilli  histologically  in  such  lesions.  This 
may  be  due  perhaps  to  the  fact  that,  owing  to  degeneration,  most  of 
the  tubercle  bacilli  have  lost  their  acid-fastness.  If  tubercles  heal,  as 
they  often  do,  they  undergo  a  fibrinous  change,  are  surrounded  by  con- 
nective tissue  and,  if  central  necrosis  has  begun  at  the  time  that  healing 
sets  in,  calcification  results.  For  more  detailed  descriptions  we  refer 
the  reader  the  Text  books  of  Pathology  of  Adami's,  MacCallum's  or 
Delafield  and  Prudden. 

There  has  been  a  great  deal  of  discussion  concerning  the  manner  in 
which  tubercle  bacilli  enter  the  human  and  animal  body  in  the  course  of 
spontaneous  infection.  A  thorough  discussion  of  this  will  be  found  in 
the  recent  book  by  Calmette,  L' Infection  Bacillaire  de  la  Tuberculose 
(Masson,  Paris,  1920),  Chapter  8,  p.  110.  Calmette  believes  that  when 
a  tubercle  bacillus  "is  deposited  on  the  surface  of  the  skin  or  a  mucous 
membrane  or  is  introduced  into  the  healthy  body  by  another  route"  it 
becomes  the  prey  of  leucocytes  which  carry  it  into  the  lymphatic  circu- 
lation and  into  the  blood.  The  leucocytic  enzymes  are  not  capable  of 
digesting  the  organism  and  eventually  the  organism  is  deposited  in  the 
lymphatics  when  the  leucocyte  degenerates. 

Tubercle  bacilli  may  remain  latent  in  the  body,  in  lymph  nodes, 
especially,  for  long  periods.  It  appears  that  the  point  of  entrance  of  the 
tubercle  bacilli  into  the  body  may  be  through  the  tonsils,  and  secondarily 
thence  through  the  lymphatics,  then  to  other  organs.  Pulmonary  in- 
fection may  be  either  by  direct  inhalation  or  indirectly  through  the 


THE  TUBERCLE   BACILLUS  595 

lymphatics.  Calmette  believes  that  actual  direct  infection  of  the  lung 
by  inhaled  bacilli  is  relatively  rare  and  bases  this  upon  experimental 
evidence.  This,  however,  is  not  in  agreement  with  the  bulk  of  evi- 
dence, and  direct  inhalation  is  probably  the  most  common  manner  of 
invasion. 

According  to  the  researches  of  Bartel  and  many  others,  it  appears 
that  direct  infection  through  the  apparently  uninjured  mucous  mem- 
brane of  the  intestinal  tract  may  take  place,  and  after  such  entrance  the 
bacilli  may  be  carried  by  the  lymphatics  and  blood  to  the  lungs  and 
other  parts  of  the  body.  Calmette  states  that,  in  all  susceptible  ani- 
mals, man  included,  and  in  all  its  varieties  of  localization,  tuberculosis 
in  the  large  majority  of  cases  originates  in  a  primary  infection  of  the 
lymphatics  which  takes  its  origin  by  entrance  of  the  tubercle  bacilli 
through  the  mucous  membranes  of  the  digestive  tract,  chiefly  the 
mucous  membranes  of  the  mouth,  pharynx  and  intestine.  This  is  the 
extreme  view,  but  one  that  is  favored  in  addition  to  Calmette  by  Von 
Behring,  Ravenel  and  others. 

Opie  25  as  a  result  of  recent  studies,  states  that  first  infection  with 
tuberculosis  may  occur  either  by  way  of  the  lungs  or  the  gastro-intestinal 
tract,  and  the  occurrence  of  one  lesion  tends  to  prevent  the  other. 

In  man,  tuberculosis  is  most  common  in  the  lungs  where  it  usually 
starts  in  the  apices.  The  apical  situation  of  early  tubercles  is  not 
entirely  explained.  A  number  of  theories  have  been  advanced,  most  of 
them  based  on  anatomical  reasoning.  In  the  lungs  there  may  be  a 
miliary  distribution  of  tubercles  or,  by  coalescence  of  these,  large 
areas  of  consolidation  may  occur,  which  are  then  spoken  of  as  phthisis. 
Extension  to  the  pleura  is  common. 

Although  pulmonary  infections  constitute  the  very  large  majority 
of  cases  of  tuberculosis  in  adult  life,  this  is  not  strictly  true  of  childhood. 
In  statistics  quoted  by  Calmette  for  Europe,  it  was  found  by  Ham- 
berger  and  Sluka  2G  that  of  160  cases  in  children  there  were  only  50  per 
cent  pulmonary  lesions.  According  to  Dr.  Holt's  statistics  for  New 
York,  however,  of  119  autopsies  of  tuberculous  children,  pulmonary 
lesions  were  found  in  99  per  cent.  In  1515  autopsies  studied  by 
Comby  during  fifteen  years,  involvement  of  the  tracheo-bronchial 
lymph  nodes  was  found  in  all.  Aside  from  the  pulmonary  and 
lymphatic  infections,  tuberculosis  may  occur  in  practically  all  other 
parts  of  the  body. 

23  Opie,  Am.  Kev.  of  Tuberculosis,  vol.  4,  1920,  p.  629. 
26  Hamburger  and  Sluka,  cited  from  Calmette,  loc.  cit. 


5%  PATHOGENIC   MICROORGANISMS 

Tuberculosis  of  the  skin  or  lupus  is  a  common  disease.  Involve- 
ment of  the  bones  and  joints  may  occur,  and  according  to  Fraser27 
may  in  many  cases  occur  without  any  previous  tuberculosis.  Tuber- 
culous meningitis  is  not  infrequent  in  children,  and  is  always  fatal. 
Calmette  quotes  Grunberg's  studies  of  the  comparative  frequency  of 
mortality  of  children  from  tuberculosis  in  568  families.  He  analyzed 
209  deaths  by  tuberculosis  in  children  from  birth  to  fifteen  years, 
from  birth  to  one  year,  82  per  cent  were  meningitic,  6  per  cent  were 
pulmonary  and  3  per  cent  other  forms  of  tuberculosis.  At  fifteen  years, 
only  6  cases  were  meningitis,?  pulmonary  and  5  of  other  forms. 

The  liver  may  be  the  site  of  tubercles,  and  tuberculosis  of  the  spleen 
has  been  observed  though  it  is  not  particularly  common.  The  kidneys 
and  the  geni to-urinary  system  are  frequently  involved,  and  the  supre- 
renal  gland  may  be  tuberculous,  and  in  this  case  may  lead  to  a  condition 
spoken  of  as  Addison's  disease. 

In  the  intestines  themselves,  various  forms  of  tuberculosis  have  been 
described.  It  appears  that  the  only  parts  of  the  body  in  which  tuber- 
culosis is  not  common  are  the  muscle  tissues  themselves,  and  the  wall 
of  the  stomach. 

Rosenberger  28  has  reported  finding  tubercle  bacilli  in  the  circu- 
lating blood  of  all  cases  of  human  tuberculosis  which  he  examined. 
This  announcement  aroused  much  interest  and  has  led  to  many  investi- 
gations by  other  workers.  Rosenberger 's  results  were  obtained  by 
morphological  examination  of  smears  of  citrated  blood  taken  from  the 
patients,  dried  upon  slides  and  laked  with  distilled  water.  Many  other 
observers  have  failed  to  confirm  Rosenberger's  results.  Anderson 29 
examined  47  cases  in  which  tubercle  bacilli  were  found  in  the  sputum 
and  one  case  of  joint  tuberculosis.  In  none  of  these  48  cases  was  he 
able  to  obtain  tubercle  bacilli,  either  by  morphological  examination 
nor  by  guinea-pig  inoculation.  Brem30  subsequently  found  that 
laboratory  distilled  water  may  frequently  contain  acid-fast  saprophytes 
— a  fact  which  may  account  in  many  cases  for  errors  when  morphologi- 
cal examination  alone  is  relied  upon  and  blood  examined  by  the  tech- 
nique of  Rosenberger.  This,  too,  is  suggested  by  the  finding  of  acid- 
fast  bacilli  in  the  blood  of  perfectly  healthy  individuals.  Therefore, 
although  the  bacilli  may  be  present  in  the  blood  in  a  certain  number  of 

27  Fraser,  Jour.  Exper.  Med.,  No.  4,  16,  1912. 

28  Rosenberger,  Am.  Jour,  of  Med.  Sc.,  cxxxvii,  1909. 

29  Anderson,  U.  S.  P.  H.  Service,  Hygienic  Lab.,  Bull.  57,  1909. 

30  Brem,  Jour.  A.  M.  A.,  liii,  1909. 


THE  TUBERCLE   BACILLUS  597 

cases  it  does  not  seem  likely  that  they  are  so  distributed  in  anything  like 
the  high  percentages  found  by  Rosenberger.31 

Although,  therefore,  in  patients  suffering  from  tuberculosis,  the 
presence  of  the  tubercle  bacilli  in  the  blood  is  generally  slight  or  intermit- 
tent, there  may  be  times  when  large  numbers  of  tubercle  bacilli  are 
thrown  into  the  blood  stream,  and,  according  to  the  manner  and  quan- 
tity thus  distributed,  secondary  foci  or  general  miliary  tuberculosis  may 
occur. 

Bacillus  tuberculosis  (typus  humanus)  is  pathogenic  for  guinea- 
pigs,  less  markedly  for  rabbits,  and  still  less  so  for  dogs.  It  is  slightly 
pathogenic  for  cattle,  a  question  spoken  of  more  extensively  below. 

Secondary  Infection  in  Tuberculosis. — An  important  consideration 
in  the  symptomatology  and  prognosis  of  pulmonary  tuberculosis  is  the 
fact  that  on  the  basis  of  the  chronic  inflammatory  condition  of  bronchii 
and  alveoli  in  the  neighborhood  of  tuberculous  processes  in  the  bron- 
chiectatic  cavities  and  perhaps  in  cavities  communicated  with  bron- 
chioles, masses  of  bacteria  of  various  species  may  accumulate  and  habit- 
ually lodge.  Staphylococci,  streptococci,  Gram-negative  cocci,  and 
frequently  influenza  bacilli  may  be  present  in  such  cases  and  materially 
contribute  to  the  illness  of  the  patient  by  superimposing  acute  and 
subacute  inflammatory  processes  upon  the  tuberculous  one. 

FREQUENCY    AND    TRANSMISSION 

In  man,  tuberculosis  is  the  most  common  of  diseases.  Naegeli's 
statistics,  based  on  a  large  series  of  autopsies,  show  not  only  the  fre- 
quency of  the  disease,  but  its  relation  to  age.  Before  one  year  of  age 
he  finds  it  very  rare.  From  the  first  to  the  fifth  year  it  is  rare,  but 
usually  fatal.  From  the  fifth  to  the  fourteenth  year,  one-third  of  his 
cases  showed  tuberculosis;  from  the  fourteenth  to  the  eighteenth  year, 
one-half  of  the  cases.  Between  eighteen  and  thirty,  almost  all  the  cases 
examined  showed  some  trace  of  tuberculous  infection.  Three-quarters 
of  these  were  active,  one-quarter  healed.  Two-fifths  of  all  deaths  occur- 
ring at  these  ages  were  due  to  tuberculosis.  After  the  age  of  thirty, 
active  lesions  gradually  diminished,  healed  lesions  increased. 

In  1900  it  was  stated  that  the  average  yearly  mortality  from  tuber- 
culosis in  New  York  amounted  to  6000,  and  that  in  Manhattan  alone 
there  were  .constantly  20,000  tuberculous  persons.  Cornet  estimates 


31  Suzuki  and  TaJcaki,  Centralbl.  f.  Bakt.,  Ixi,  1911. 


598  PATHOGENIC   MICROORGANISMS 

that  in  1894  the  deaths  in  Germany  from  all  other  infectious  diseases 
amounted  to  116,705,  those  from  tuberculosis  alone  to  123,904.  Similar 
statistics  might  be  chosen  from  the  health  reports  of  any  large  city. 
While  the  disease  is  less  common  in  rural  districts  than  in  large  towns, 
the  difference  is  not  so  striking  as  is  generally  supposed. 

Kober  32  states  that,  owing  to  active  measures  of  prevention,  the 
death  rate  from  tuberculosis  has  been  reduced  from  326  per  100,000  in 
1888  to  147.6  in  1913,  which  he  says  means  that,  if  the  former  rate  of 
mortality  had  been  continued  "the  number  of  deaths  from  this  disease 
last  year  (1914)  would  have  been  322,027,  instead  of  143,000,"  meaning 
the  saving  of  over  179,000  lives. 

Although  there  has  been  much  discussion  concerning  the  different 
methods  of  infection,  there  seems  to  be  very  little  doubt  at  the  present 
time  that  inhalation  is  the  most  common  means  of  human  infection. 
In  coughing,  expectoration  and  sneezing,  small  droplets  of  fluid  in 
which  all  kinds  of  microorganisms  are  found,  are  sprayed  into  the  air, 
and  these  may  be  deposited  upon  the  mucous  membranes  of  people 
in  close  contact  with  the  disease.  The  striking  distance  of  such  droplet 
infection  is  not  very  large,  but  is,  as  Kober  points  out,  particularly 
dangerous  because  the  bacilli  thus  enter  the  respiratory  passages 
directly  from  body  to  body.  In  addition  to  this,  the  tubercle  bacilli 
may  remain  alive  in  dust  sufficiently  dry  to  be  blown  about  by  draughts 
and  winds.  Although  the  bacilli  are  not  spore  bearers,  their  acid-fast 
nature  renders  them  somewhat  more  resistant  to  desiccation  and  sun- 
light than  are  most  other  germs. 

Next  to  direct  inhalation,  the  most  frequent  method  of  transmission 
is  probably  through  the  digestive  tract.  Such  infection  may  take  place 
by  direct  contamination  of  food  from  the  expectoration  and  saliva  of 
consumptives  or  by  indirect  infection  of  food,  and  milk,  through  the 
agency  of  fingers,  flies,  etc. 

Milk  Infection. — The  question  of  intestinal  infection,  however,  is 
particularly  important  in  connection  with  transmission  of  bovine  tuber- 
culosis to  man  through  the  agency  of  infected  milk.  The  general  public 
has  probably  very  little  idea  of  the  frequency  of  tuberculosis  in  cattle. 
In  a  community  supervised  more  closely  than  usual,  the  work  of  Public 
Health  Service  bacteriologists  in  Washington,  revealed  6.72  per  cent  of 
samples  of  market  milk  infected  with  tubercle  bacilli.  This  percentage 
is  probably  very  much  lower  than  that  which  would  naturally  be  found 
in  districts  with  a  less  well-developed  dairy  supervision,  and  in  some 

82  Kober,  Kep.  No.  309,  U.  S.  P.  H.  S.  Keports,  October,  1915. 


THE   TUBERCLE   BACILLUS 


599 


of  the  poorer  farm  districts  of  the  country  the  cattle  tuberculosis  situa- 
tion is  actually  appalling.  The  question  of  how  frequently  infection 
with  bovine  tubercle  bacilli  through  the  intestinal  tract  .may  occur 
is  still  a  matter  of  some  controversy. 

V.  Behring  expressed  the  belief  that  a  large  percentage  of  all  cases 
of  tuberculosis  originated  in  childhood  from  infection  through  the  in- 
testinal tract.  He  determined  that  tubercle  bacilli  may  penetrate  the 
intestinal  mucosa  without  causing  lesions.  Behring's  contention  caused 
a  great  deal  of  discussion,  and  the  question  he  raised  is  intimately 
bound  up  with  the  problem  of  the  virulence  of  bovine  tubercle  bacilli 
for  human  beings,  as  he  assumes  that  the  infection  is  due  to  the  use  of 
infected  milk, 

COMBINED  TABULATION,  CASES    REPORTED  AND  OWN  SERIES  OF 

CASES 

(From  Park  and  Krumwiede,  loc.  cit.) 


Diagnosis. 

Adults,  16  Years 
and  Over. 

Children, 
5  to  16  Years. 

Children 
Under  5  Years. 

Human. 

Bovine 

Human 

Bovine. 

Human. 

Bovine. 

Pulmonarv  tuberculosis 

568 
2 
22 
15 

6 

28 

4 

18 
11 
1 

2 

1? 
1 

3    ' 
1 

1 
1 

1 

11 

4 
33 

7 

2 

4 

1 

7 

2 
26 
1 
1 

20 

7 

3 
1 

1 

12 
2 
15 
6 

13 

28 

3 

45 
14 
21 

1 
1 

20 

13 

10 
5 

8   ' 

1 
2 

Tuberculous  adenitis,  axillary  or  inguinal 
Tuberculous  adenitis,  cervical  
Abdominal  tuberculosis  .  .  .  
Generalized  tuberculosis,  alimentary  ori- 
gin 

Generalized  tuberculosis 

Generalized  tuberculosis,  including  men- 
inges,  alimentary  origin  
Generalized  tuberculosis,  including  men- 
inges 

Tuberculous  meningitis  
Tuberculosis  of  bones  and  joints  
Genito-urinary  tuberculosis 

Tuberculosis  of  skin            t 

Miscellaneous  Cases 
Tuberculosis  of  tonsils  
Tuberculosis  of  mouth  and  cervical  nodes 
Tuberculous  sinus  or  abscesses 

Sepsis,  latent  bacilli  

Totals  

677 

9 

99 

33 

161 

59 

Mixed  or  double  infections,  4  cases. 


600  PATHOGENIC   MICROORGANISMS 

The  problem  is  plainly  of  the  greatest  importance,  and  for  this 
reason  has  been  diligently  investigated  during  the  last  few  years.  The 
only  reliable  method  of  approaching  it  has  been  to  isolate  the  tubercle 
bacilli  from  diseased  human  beings  and  determine  for  each  case  whether 
the  organism  obtained  belonged  to  the  human  or  bovine  type.  These 
types  can  be  differentiated  definitely  by  cultural  characteristics  and 
pathogenicity,  and  it  is  not  likely  that  the  type  changes  during  the 
sojourn  in  the  human  body.  Granted  this  permanence  of  tyre,  it  is 
naturally  of  much  value  in  revealing  the  source  of  an  infection,  to  deter- 
mine whether  or  not  a  human  being  is  harboring  a  bacillus  of  the  human 
type  or  one  of  the  bovine  type. 

One  of  the  most  valuable  contributions  made  to  this  problem  during 
the  last  three  years  is  that  of  Park  and  Krumwiede.33  The  above 
tabulation  is  taken  from  their  paper  and  represents  a  summary  of  their 
own  cases  and  those  reported  by  others. 

From  this  table  it  is  evident  that  out  of  a  total  of  1042  cases,  101 
only  were  bovine  in  origin  and  over  50  per  cent  of  these  occurred  in 
children  under  five  years  of  age.  Fifty-one  out  of  the  59  cases  occurring 
in  the  161  infants  were  directly  or  indirectly  traced  to  alimentary  infec- 
tion. 

It  seems  reasonably  accurate,  therefore,  to  state  the  case  as  follows: 
Human  adults  are  relatively  insusceptible  to  bovine  infection.  Such 
infection  can  take  place,  but  is  unusual.  Below  sixteen  years  of  age  the 
human  race  is  relatively  more  susceptible  and  up  to  this  age  the  danger 
of  milk  infection  is  unquestionably  great,  this  source  accounting  for 
about  one-third  of  the  cases.  Below  five  years  the  danger  is  greatest. 
This  table  alone  should  form  sufficient  evidence  to  silence  absolutely 
any  doubts  as  to  the  dangers  of  milk  infection  and  remove  any  objections 
to  the  most  rigid  sanitary  control  of  milk  supplies. 

On  the  other  hand,  it  also  shows  that  Behring's  original  claims  were 
far  too  sweeping  and  can  not  be  upheld. 

Kober  34  also  calls  attention  to  the  fact  that  one  must  not  be  deceived 
into  believing  that  childhood  is  the  only  really  dangerous  age  for  infec- 
tion with  tuberculosis,  and  quotes  the  results  of  a  French  committee 
which,  in  a  small  group  carefully  investigated,  found  64  cases  in  which 
the  disease  was  transmitted  from  husband  to  wife,  43  cases  in  which  it 
was  transmitted  from  wife  to  husband,  38  cases  transmitted  from  brother 

R3  Park  and  Krumwiede,  Jour,  of  Med.  Ees.,  Oct.,  1910. 

31  Kober,  Repr.  No.  309,  TJ.  S.  P.  H.  S.  Reports,  October,  1915. 


THE  TUBERCLE  BACILLUS  601 

to  sister,  19  from  mother  to  child,  16  from  other  relatives,  and  in  33  cases 
it  was  traced  to  people  who  were  not  relations,  but  with  whom  the 
patient  had  been  in  communication.  He  also  quotes  Zasetsky  who 
reports  the  case  of  a  tuberculous  woman  who,  in  the  course  of  eleven 
years  married  three  husbands  who  had  been  previously  healthy.  The 
first  one  died  of  tuberculosis  seven  years  after  marriage,  the  second  three 
years  later,  and  the  third  at  the  time  of  the  report  had  the  disease,  the 
wife  in  the  meantime  having  died  of  tuberculosis. 

The  fact  that  tubercle  bacilli  may  be  conveyed  in  dust  has  been 
indicated  above,  but  there  are  other  means  by  which  habitual 
inhalation  of  dust  may  favor  the  spread  of  tuberculosis,  namely, 
by  virtue  of  the  irritant  properties  of  inhaled  dust  in  predisposing 
the  lung  to  infection.  Attention  to  the  dangers  of  trades  in  which 
dust  is  an  habitual  environmental  factor  has  been  particularly  empha- 
sized by  Winslow  and  Greenberg.  Sommerfeld,  whom  we  quote  from 
Kober,  made  a  statistical  study  in  which  he  showed  that  in  the 
population  of  Berlin,  the  average  tuberculosis  death  rate  was  4.93 
per  1000.  The  rate  in  the  non-dusty  trades,  was  2.39,  and  in  the 
dusty  trades  5.2.  He  also  states  that  the  analysis  of  tuberculosis  in 
the  towns  in  Vermont  where  granite  and  marble  cutting  is  carried 
out  showed  a  tuberculosis  rate  of  2.2  per  1000  against  a  rate  of  1.3 
for  the  whole  state,  and  Ropke  is  stated  by  the  same  writer  to  have 
shown  that  the  mortality  from  tuberculosis  of  the  population  in  the 
large  cutlery  center,  Solingen,  in  Germany  was  reduced  from  5.4 
per  1000  in  1885,  to  1.8  in  1910,  by  measures  aimed  at  the  control 
of  the  dust  in  work  rooms.  A  recent  study  by  Drury 35  shows  that 
polishers  and  grinders  in  axe  factories  are  subject  to  a  death  rate  from 
pulmonary  tuberculosis  considerably  above  that  of  others  in  the  same 
mill.  In  the  two  decades  from  1900  to  1919,  the  polishers  and  grinders 
showed  a  death  rate  of  19  per  1000  as  against  6.5  of  the  entire  mill 
population,  and  between  1  and  2.4  of  the  general  population  of  the 
district. 

In  regard  to  the  predisposing  factors  to  tuberculous  infection,  many 
phases  enter  into  the  problem  in  this  disease  which  exert  a  much  less 
direct  or  perhaps  negligible  influence  in  connection  with  other  infections. 
There  can  be  no  question  about  the  fact  that  poverty,  with  its  coincident 
crowding  in  living  quarters,  close  personal  contact  at  night,  insufficient 
warmth,  and  particularly  undernutrition  and  low  fat  diet,  play  a  role 


35  Drury,  Pub.  Health  Reeports,  U.  S.  P.  H.  S.,  Feb.,  1921,  Vol.  36,  No.  5. 


602  PATHOGENIC   MICROORGANISMS 

of  immense  importance  in  tuberculosis.  In  no  disease  is  prevention  so 
intimately  influenced  by  general  sociological  and  economic  improve- 
ment as  in  tuberculosis. 

Wernicke  36  in  a  study  of  the  relationship  of  diseases  to  social 
conditions  shows  an  almost  direct  relationship  between  the  provision 
of  air  space  and  parks  in  cities  to  tuberculosis.  The  statistics  of  the 
influence  of  the  war  upon  tuberculosis  have  not  yet  become  avail- 
able for  study,  but  it  is  important  to  note  that  at  the  present  writing 
we  are  informed  by  sanitarians  who  have  returned  from  Europe  that  the 
sanitary  problem  in  the  European  States  is  very  largely  one  of  tuber- 
culosis, and  that  the  effects  of  prolonged  undernutrition,  especially 
upon  children  during  the  war  years  has  resulted  in  an  enormously 
increased  tuberculosis  rate. 

The  question  of  the  inheritance  of  tuberculosis  has  frequently  been 
raised,  and  a  large  literature  on  this  subject  has  accumulated,  but  an 
analysis  of  this  literature  seems  to  show  that  inheritance  must  be 
regarded  as  predisposition  rather  than  as  a  method  of  direct  infection. 
Children  of  tuberculous  parents  are  likely  to  be  more  susceptible  to 
tuberculosis  and,  of  course,  are  expos3d  to  tuberculosis  more  intimately 
during  the  early  years  of  life  than  are  children  of  normal  parents. 
There  is  no  direct  proof  that  tuberculosis  is  transmitted  from  mother  to 
the  foatus. 

Prevention. — As  to  preventive  measures,  we  must  refer  the  reader 
to  special  books  on  the  subject  since  the  problem  is  too  large  to  be 
dealt  with  briefly  with  anything  like  completeness.  The  following 
summary  of  preventive  measures  is  based  largely  upon  the  conclusions 
reached  in  Kober's  discussion  of  this  subject: 

We  may  assume,  as  premises  for  prevention,  that  tuberculosis  can  be 
transmitted  at  all  periods  of  life  and  that  foci  acquired  in  youth  may 
be  arrested  but  light  up  under  conditions  of  general  undernutrition, 
malnutrition,  etc.,  etc.,  in  later  years.  Infection  may  be  direct  from 
person  to  person,  indirect  through  contaminated  food,  fomites,  flies; 
through  dust,  and  in  childhood  through  milk  from  infected  cattle. 
The  most  common  manner  of  acquiring  tuberculosis  is  by  inhalation 
and,  next  to  that,  probably  through  the  digestive  tract. 

The  most  important  factor  in  the  prevention  of  tuberculosis  is 
education.  This  must  elucidate  the  method  of  infection  and  the  im- 
portance of  the  economic  and  sociological  factors  as  they  effect  habits 
of  food,  sleep  and  fresh  air. 

36  Wernicke,  quoted  from  Kober. 


THE  TUBERCLE  BACILLUS  603 

• 

Direct  infection  must  be  prevented  by  compulsory  notification, 
care  and  disinfection  of  expectorations,  isolation,  at  least  as  far  as 
the  possibilities  of  sputum  infection  are  concerned,  in  the  home,  in 
hospitals,  etc.,  with  introduction  of  pocket  sputum  flasks  and  the  other 
simple  measures  by  which  a  well-controlled  tuberculosis  patient  can 
avoid  infecting  others.  The  actual  prevention  in  deed,  as  well  as  word, 
of  expectoration  in  public  places,  the  protection  of  public  drinking  places, 
introduction  of  individual  cups  and  public  cleanliness  in  general;  espe- 
cial supervision  of  these  conditions  in  places  of  public  lodgment  and 
public  amusement  in  schools  and  public  conveyances;  introduction 
of  vacuum  cleaning,  etc. 

Attention  in  regard  to  the  marriage  of  tuberculous  people. 

Supervision  of  dairies  and  the  marketing  of  milk  by  governmental 
grading,  and  control,  with  pasteurization  of  suspicious  milk  or  milk  not 
produced  under  the  required  conditions  for  grade  "A"  milk. 

Public  provision  for  the  proper  and  humane  care  of  tuberculous 
people  in  state  and  municipal  sanatoria  so  arranged  that  the  poor  will 
regard  them  as  havens  of  hope,  rather  than  as  penalties  imposed  for 
disease. 

The  public  must  be  educated  in  knowing  that  tuberculosis  is  a  cu- 
rable disease,  provided  that  the  diagnosis  is  made  early  and  clinical 
facilities  must  be  so  arranged  in  cities  so  that  accurate  diagnosis  in  the 
early  stages  may  be  made  and  proper  fresh  air  and  nutritional  care 
instituted  if  necessary,  at  public  expense.  In  our  cities,  roofs,  play- 
grounds, parks,  etc.,  should  be  provided  for  school  children.  Summer 
care  of  children  living  in  the  crowded  districts  must  be  developed  on  a 
more  generous  and  more  important  scale.  The  nutrition  of  school 
children  in  the  public  schools  must  be  supervised  and  subsidized  so 
that  no  child  in  a  civilized  community  should  suffer  at  any  time  from 
under  nutrition. 

The  prevention  of  tuberculosis  is  only  in  small  part  a  medical  prob- 
lem and  must  rest  in  its  last  analysis  on  the  prevention  of  the  means  of 
direct  infection  and  the  predisposing  considerations  in  which  the  sociol- 
ogist and  the  educator  must  play  as  important  a  part  as  the  physician. 

Chemical  Analysis  of  Tubercle  Bacilli.37 — Diligent  efforts  by  many 
investigators  to  isolate  the  specific  toxins  which  lend  tubercle  bacilli 
their  pathogenic  properties  have  led  to  careful  chemical  analysis  of  the 
organisms.  About  85.9  per  cent  of  the  bacillus  consists  of  water;  20 

37  Hammerschlag,  Cent.  f.  klin.  Med.,  1891;  Weyl,  Deut.  med.  Woch.,  1891; 
De'Schweinits  and  Dorset,  Jour.  Amer.  Chem.  Soc.,  1895;  Hammerschlag,  loc.  cit. 


604  PATHOGENIC  MICROORGANISMS 

to  26  per  cent  of  the  residue  can  be  extracted  with  ether  and  alcohol. 
This  material  consists  of  fatty  acids  and  waxy  substances  (fatty  acids 
in  combination  with  the  higher  alcohols).  The  residue  after  alcohol- 
ether  extraction  is  composed  chiefly  of  nitrogenous  constituents.  These 
can  be  extracted  with  dilute  alkaline  solutions,  and  consist  chiefly  of 
nucleoproteins.  After  removal  of  the  so-called  nucleoproteins — that 
is  the  material  which  comes  down  on  treatment  with  dilute  acetic  acid 
in  the  cold,  there  remains  a  small  amount  of  coagulable  protein  and, 
we  have  recently  observed,  small  amounts  of  a  substance  that  reacts 
like  Bence-Jones  protein.38  The  final  residue  contains  alcohol  precip- 
itable  substances — proteoses  and  polypeptids  that  constitute  the  active 
substances  of  the  tuberculin  reactions.  Cellulose  is  also  found  and  is 
supposed  to  represent  the  framework  of  the  cell  membrane,  and  there  is 
an  ash  rich  in  calcium  and  magnesium. 

Toxins  of  the  Tubercle  Bacillus. — THE  TUBERCULINS. — Filtrates  of 
bouillon  cultures  of  Bacillus  tuberculosis39  will  occasionally  produce 
slight  emaciation  when  injected  into  guinea-pigs,  and  when  adminis- 
tered to  tuberculous  subjects  in  sufficient  quantity  will  give  rise  to 
marked  increase  of  temperature.  It  is  likely,  therefore,  that  the 
tubercle  bacillus  actually  secretes  a  soluble  toxin.40 

The  chief  toxic  principles,  however,  of  Bacillus  tuberculosis  are 
probably  endotoxins  or  bacterial  proteins,  bound  during  cell  life  to  the 
body  of  the  bacillus.  Dead  bacilli  will  produce  sterile  abscesses  when 
inj ected  into  animals.  Prudden  and  Hodenpyl,41  Straus  and  Gamaleia,42 
and  others,43  moreover,  have  shown  that  the  injection  of  dead  and  care- 
fully washed  cultures  of  this  bacillus  will  produce  lesions  histologi- 
cally  similar  to  those  occurring  after  infection  with  the  living  germs, 
and  will  often  lead  to  marasmus  and  other  systemic  symptoms  of 
poisoning. 

The  hope  of  actively  immunizing  with  substances  obtained  from 
dead  bacilli  led  Koch  to  employ  various  methods  of  extraction  of  cul- 
tures for  the  manufacture  of  tuberculin. 

"Old  Tuberculin"**  (Koch)  ("T.A.K.").— The  first  tuberculin  made 


88  Zinsser,  Journ.    exp.  med.,  Nov.,  1921. 

39  Straus  and  Gamaleia,  Arch.  med.  exp.,  1891. 

40  Denys,  '  <  Le  Bouillon  Filtre, ' '  Louvain,  1905. 

41  Prudden  and  Hodenpyl,  N.  Y.  Med.  Jour.,  June,  1891;  Prudden,  ibid.,  Dec.  5. 

42  Straus  and  Gamaleia,  loc.  cit. 
43Mafucci,  Cent.  f.  allg.  Path.,  1890. 

44  Koch,  Cent.  f.  Bakt.,  1890;  Deut.  med.  Woch.,  1891. 


THE  TUBERCLE   BACILLUS  605 

by  Koch  is  produced  in  the  following  manner:  Tubercle  bacilli  are 
grown  in  slightly  alkaline  5  per  cent  glycerin-pepton  bouillon  for  six  to 
eight  weeks.  At  the  end  of  this  time,  growth  ceases  and  the  corrugated 
pellicle  of  tubercle  bacilli,  which  during  growth  has  floated  on  the 
surface,  begins,  here  and  there,  to  sink  to  the  bottom.  The  entire 
culture  is  then  heated  on  a  water-bath  at  about  80°  C.,  until  reduced  to 
one-tenth  of  its  original  volume.  It  is  then  filtered  either  through 
sterile  filter  paper  or  through  porcelain  filters.  The  resulting  filtrate  is 
a  rich  brown,  syrupy  fluid,  containing  the  elements  of  the  original  cul- 
ture medium  and  a  50  per  cent  glycerin  extract  of  the  tubercle  bacilli. 
While  the  glycerin  is  of  sufficient  concentration  to  preserve  it  indef- 
initely, 0.5  per  cent  phenol  may  be  added  as  an  additional  precaution. 
Dilutions  of  this  fluid  are  used  for  diagnostic  and  therapeutic  purposes. 

"New  Tuberculin"*5  (Koch)  (TA,  TO,  TR).— Koch  believed  that 
the  immunity  resulting  from  treatment  with  the  old  tuberculin  was 
purely  an  antitoxic  immunity,  devoid  of  all  antibacterial  action.  The 
use  of  whole  dead  tubercle  bacilli  for  immunization  purposes,  however, 
was  impracticable;  because,  injected  subcutaneously,  they  were  not 
absorbed,  and  introduced  intravenously  they  were  deposited  in  the  lungs 
and  gave  rise  to  lesions.  Koch  was  led,  therefore,  to  resort  to  more 
energetic  extraction  of  the  bacilli  in  the  hope  of  procuring  a  substance 
which  could  be  easily  absorbed  and  would  at  the  same  time  give  rise, 
when  injected,  to  antibodies  more  definitely  bactericidal.  By  extract- 
ing tubercle  bacilli  with  decinormal  NaOH,  for  three  days,  filtering 
through  paper  and  neutralizing,  he  obtained  his  TA  (alkaline  tubercu- 
lin). This  preparation  seemed  to  fulfill  some  of  the  hopes  of  its  dis- 
coverer, but  had  the  disadvantage  of  often  producing  abscesses  at  the 
points  of  injection.  Koch  then  resorted  to  mechanical  trituration  of 
the  bacilli.  The  method  he  subsequently  followed  for  tuberculin  pro- 
duction is  now  extensively  used,  and  is  carried  out  as  follows:46 

Virulent  cultures  of  tubercle  bacilli  are  dried  in  vacuo  and  thor- 
oughly ground  in  a  mortar.  Grinding  is  continued  until  stained  prep- 
arations reveal  no  intact  bacilli.  (This  is  done  by  machinery  in  all  large 
manufactories.)  One  gram  of  the  dry  mass  is  shaken  up  in  100  c.c.  of 
sterile  distilled  water.  This  mixture  is  then  centrifugalized  at  high 
speed.  The  supernatant  fluid,  known  as  TO  (Tuberculin-Oberschicht), 
contains  the  water-soluble  constituents  of  the  bacillus,  gives  no  precip- 


45  Koch,  Deut.  med.  Woch.,  14,  1897. 
"Ruppel,  Lancet,  March  28,  1908. 


606  PATHOGENIC   MICROORGANISMS 

itate  on  the  addition  of  50  per  cent  glycerin,  and  has  the  same  physiolog- 
ical action  as  the  old  tuberculin.  The  residue  TR  (Tuberculin-Ruck- 
stand),  after  pouring  off  TO,  is  again  dried  and  ground  up,  and  again 
shaken  in  water  and  centrifugalized.  This  process  is  repeated  several 
times,  and  eventually,  after  three  or  four  repetitions,  all  the  TR  goes  into 
emulsion.  The  total  volume  of  water  used  for  these  TR  extractions 
should  not  exceed  100  c.c.  All  of  the  TR  emulsions  are  then  mixed 
together.  This  gives  TR  a  precipitate  with  50  per  cent  of  glycerin,  and 
is  supposed  by  Koch  to  contain  substances  important  in  producing  an 
antibacterial  immunity.  For  purposes  of  standardization,  the  amount 
of  solid  substance  in  5  c.c.  of  the  TR  is  determined  by  evaporation  in 
vacuo  and  drying.  To  the  rest  are  added  a  little  glycerin  and  formalde- 
hyd  and  enough  water  to  allow  each  cubic  centimeter  of  the  solution 
to  contain  0.002  gram  of  solid  material.  Thus  the  culture  and  the 
medium  remaining  the  same,  fairly  accurate  standardization  is  possible. 

"New  Tuberculin-bacillary  Emulsion."47 — In  1901,  Koch  combined 
"TO"  and  "TR"  by  putting  forth  a  preparation  referred  to  as  "Bazil- 
lenemulsion."  This  consists  of  an  emulsion  of  pulverized  bacilli 
1  :  100  in  distilled  water.  After  several  days  of  sedimentation  to 
remove  the  coarser  particles,  the  supernatant  fluid  is  poured  off  and 
fifty  per  cent  volume  of  glycerin  is  added  to  it  for  purposes  of  preserva- 
tion. This  preparation  contains  5  milligrams  of  solid  substance  in  each 
cubic  centimeter. 

Bouillon  F litre  (Denys)48 — This  preparation  consists  of  the  filtrate 
(through  Chamberland  filters)  of  5  per  cent  glycerin-pepton-bouillon 
cultures  of  Bacillus  tuberculosis.  Phenol  0.25  per  cent  is  added  to 
insure  sterility.  The  filtered  bouillon  corresponds  to  the  unconcen- 
trated  old  tuberculin  of  Koch,  but,  not  having  been  heated,  is  supposed 
by  Denys  to  contain  important  soluble  and  possibly  thermolabile 
secretory  products  of  the  bacillus. 

Tuber culoplasmin  (Buchner  and  Hahn)49 — Buchner  and  Hahn,  by 
crushing  tubercle  bacilli  by  subjecting  them  to  a  pressure  of  400  atmos- 
pheres, obtained  a  cell-juice  in  the  form  of  an  amber  fluid,  to  which  they 
attributed  qualities  closely  analogous  to  those  of  TR. 

Other  tuberculins  are    those  of    Beraneck,50  highly  recommended 


"7  Koch,  Dent.  mod.  Woc.h.,  1901. 

48  Dei\ys,  '  'Le  Bouillon   Filtre,  "   Lonvain,   1905. 

49  Buchner  mid   llahn,  Miinch.  med.  Woch.,  1897;   Halm,  ibid. 

50  Beraneck,  Compt.  rend,  de  1'acad.  des.  sci.,  1903. 


THE   TUBERCLE   BACILLUS  607 

clinically  by  Sahli,51  that  of  Klebs,5-  and  the  tuberculin  produced  from 
bovine  tubercle  bacilli  by  Speiigleiv™ 

Diagnostic  Use  of  Tuberculin. — Subculancous*Usc. — The  prepara- 
tion usually  employed  for  diagnostic  purposes  is  Koch's  "Old  Tubercu- 
lin" ( Alttuberculin) .  This  preparation  is  administered  by  hypodermic 
injection  of  small  quantities  obtained  by  means  of  dilutions.  The 
dilutions  are  best  made  with  a  0.5  per  cent  aqueous  carbolic  acid  solution. 
In  practice  a  1  per  cent  solution  is  made  by  pipetting  0.1  c.c.  of  tuber- 
culin into  9.9  c.c.  of  the  0.5  per  cent  carbolic  solution.  A  cubic  centi- 
meter of  this  then  contains  0.01  c.c.  of  tuberculin.  One  cubic  centi- 
meter of  this  solution  added  to  9  c.c.  of  0.5  per  cent  carbolic  acid  gives  a 
solution  in  which  each  cubic  centimeter  contains  0.001  c.c.,  or  1  mm.  of 
tuberculin. 

The  initial  dosage  in  adults  in  Koch's  54  early  work,  and  as  used  by 
Beck55  on  a  large  number  of  patients,  was  1  mgm.  This,  according  to 
present  opinions,  is  too  high,  and  most  clinicians  to-day  prefer  0.1 
to  0.2  of  a  milligram.  If  after  three  or  four  days  no  reaction  has 
occurred,  a  second  dose  of  1  mm.  is  given. 

The  reaction  itself  is  recognized  chiefly  by  the  changes  in  tem- 
perature. In  a  positive  reaction  the  patient's  temperature  will  begin 
to  increase  within  six  to  eight  hours  after  injection,  rising  sharply 
within  a  few  hours  to  0.5  or  1.5°  higher  than  the  temperature  before 
injection.  It  then  sinks  more  gradually  than  it  rose,  the  reaction  usually 
being  complete  within  thirty  to  thirty-six  hours.  With  the  tempera- 
ture there  maybe  nausea,  a  chill,  rapid  pulse,  and  general  malaise. 
Locally  visible  tuberculous  processes,  such  as  lupus,  lymph  nodes,  etc., 
may  become  tender  or  swollen,  and  if  the  tuberculosis  is  pulmonary, 
there  may  be  coughing  and  increased  expectoration.  The  temper- 
atures of  persons  subjected  to  the  test  should  be  taken  regularly  for 
three  or  four  days  before  tuberculin  is  used. 

Ophthalmo-tuberculin  Reaction. — Wolff-Eisner  56  and,  soon  after  him, 
Calmette,57  proposed  a  method  of  using  tuberculin  for  diagnostic 
purposes  by  instillation  into  the  conjunctival  sac.  In  tuberculous 


51  Sahli,  Corrbl.  d.  Schw.  Aerzte,  1906. 

tiKlebs,  Cent.  f.  Bakt%  1896;  Dent.  med.  Woch.,  1907. 

53  Spengler,  Deut.  med.  Woch.,  xxxi,  1904 ;  xxxi  and  xxxiv,  1905. 

M  Koch,  Deut.  med.  Woch.,  1890. 

55J5ecfr,  Deut.  med.  Woch.,  1899. 

66  Wolff-Eisner,  Berl.  med.  Gesell.,  May  15,  1907. 

57  Calmette,  Acad.  des  sci.,  June  17,  1907. 


008  PATHOGENIC   MICROORGANISMS 

patients  this  process  is  followed  by  a  sharp  conjunctival  congestion 
lasting  from  one  to  several  days. 

The  preparation  used  for  this  purpose  is  produced  in  the  following 
way: 

"Old  Tuberculin"  is  treated  with  double  its  volume  of  95  percent 
alcohol,  the  precipitate  allowed  to  settle  and  the  alcohol  then  filtered 
off  through  paper.  The  sediment  is  washed  with  70  per  cent  alcohol 
until  the  filtrate  runs  clear,  then  pressed  between  layers  of  filter 
paper  to  remove  excess  of  moisture,  scraped  into  a  dish,  dried  in 
vacuo  over  H^SCU,  and  broken  up  in  a  mortar  under  a  hood. 

Solutions  of  the  powder  are  made  in  sterile  normal  salt  solution,  1 
per  cent  by  weight,  boiled  and  filtered.  The  solutions  are  used  in 
strengths  of  0.5  to  1  per  cent,  a  drop  of  which  is  instilled  into  the  con- 
junctival sac.58 

Cutaneous  Tuberculin  Reaction. — Von  Pirquet  59  has  suggested  the 
cutaneous  use  of  tuberculin  for  diagnostic  purposes.  A  25  per  cent 
solution  of  "Old  Tuberculin"  was  first  used.  At  present  the  undi- 
luted substance  is  employed. 

After  sterilization  of  the  patient's  forearm,  two  drops  of  this  solu- 
tion are  placed  upon  the  skin  about  6  cm.  apart.  Within  each  of  these 
drops  scarification  is  done,  and  the  skin  between  them  scarificed  as  a 
control.  Within  twenty-four  to  forty-eight  hours,  in  tuberculous 
patients,  erythema,  small  papules,  and  herpetiform  vesicles  will  appear. 
According  to  recent  investigations,  about  70  per  cent  of  adults  show  a 
positive  reaction.  This  diminishes  its  diagnostic  value  for  adults. 

Moro  60  has  modified  this  by  making  a  50  per  cent  ointment  of 
tuberculin  in  lanolin  and  rubbing  it  into  the  skin  without  scarification. 

Complement  Fixation  in  Tuberculosis.61 — The  problem  of  comple- 
ment fixation  for  diagnostic  purposes  in  tuberculosis  has  been  very 
actively  investigated  of  recent  years.  The  most  promising  results  have 
been  reported  with  an  antigen  made  by  Besredka  of  a  filtrate  of  an 
egg-meat-broth,  upon  which  the  tubercle  bacilli  had  been  grown  for 
several  weeks  with  a  similar  filtrate  of  cultures  on  a  watery  extract  of 
potato  with  glycerin,  used  by  Petroff,  and  with  an  antigen  made  by 
Miller  and  Zinsser  by  triturating  dead  tubercle  bacilli  with  dry  crystals 

58  The  conjunctival  test  is  not  in  general  use  at  the  present  time  owing  to  pos- 
sible dangers  to  the  eyes. 

m-v.  Pirquet,  Berl.  Win.  Woch.,  xx,  1907;   Med.  Klinik,  xl,  1907. 

69  Moro,  Munch,  med.  Woch.,  1906,  p.  216. 

61  H.  E.  Miller,  Jour.  Lab.  &  Clin.  Med.,  1916,  i,  816. 


THE  TUBERCLE  BACILLUS  609 

of  NaCl  and  adding  distilled  water  to  isotonicity.  Craig,  Bronfenbren- 
ner  and  the  above-named  writers  have  reported  good  results  with 
these  various  antigens,  and,  although  it  is  too  early  to  say  which  will 
prove  most  useful,  it  is  clear  that  complement  fixation  methods  can  aid 
in  the  diagnosis  of  active  tuberculosis.  We  can,  of  course,  judge  con- 
cisely only  of  the  method  used  in  our  laboratory,  where  Miller  has 
followed  carefully  a  considerable  number  of  cases  on  which  the  method 
has  been  used.  It  would  appear  at  present  that  about  70  per  cent 
of  the  fixation  results  correspond  with  clinical  findings. 

The  Tuberculin  Test  as  Applied  to  Cattle. — In  cattle,  the  symptoms 
of  tuberculosis  are  not  easily  detected  by  methods  of  physical  diag- 
nosis until  the  disease  has  reached  an  advanced  stage.  In  consequence, 
cows  may  be  elements  of  danger  without  appearing  in  any  way  diseased. 
In  consequence,  routine  examination  of  herds  by  the  tuberculin  test  has 
become  one  of  the  necessary  measures  of  sanitation.  According  to 
Mohler,62  an  accurate  diagnosis  may  be  established  in  at  least  97  per 
cent  of  the  cases.  It  is  natural  that  a  good  deal  of  objection  to  the 
test  is  encountered  on  the  part  of  dairy  farmers  and  cattle  raisers,  and 
it  has  been  claimed  that  the  cattle  are  injured  by  the  test.  There  is,  how- 
ever, no  scientific  basis  for  this  belief,  if  the  test  is  carried  out  carefully  and 
intelligently.  As  a  matter  of  fact,  the  systematic  use  of  the  test  would 
eventually  be  distinctly  advantageous  to  the  owners  of  the  cattle  them- 
selves, since  it  has  been  shown  that  cows,  even  in  the  early  stages  of  the 
disease,  may  expel  tubercle  bacilli,  either  during  respiration  or  with  the 
feces,  and  thus  become  a  menace  to  healthy  members  of  the  herd. 

The  tuberculin  test  on  cattle  should  be  made  as  follows:  (The 
directions  given  below  are  taken  directly  from  the  circular  sent  out.  from 
the  Bureau  of  Animal  Industry  at  Washington.) 

1.  Begin  to  take  the  rectal  temperature  at  6  A.M.,  and  take  it  very 
two  hours  thereafter  until  midnight. 

2.  Make  the  injection  at  midnight. 

3.  Begin  to  take  the  temperature  next  morning  at  6  A.M.,  and  con- 
tinue as  on  preceding  day. 

To  those  who  have  large  herds  to  examine,  or  are  unable  to  give  the 
time  required  by  the  above  directions,  the  following  shortened  course  is 
recommended: 

1.  Begin  to  take  the  temperature  at  8  A.M.,  and  continue  every  2 
hours  until  10  P.M,  (omitting  at  8  P.M.,  if  more  convenient) ;  or  take  the 
temperature  at  8  A.M.,  12  M.,  and  10  P.M. 

«  Mohler,  Pub.  H.  and  Mar.  Hosp.  Serv.  Bull.,  41,  1908. 


610  PATHOGENIC  MICROORGANISMS 

2.  Make  the  injection  at  10  P.M. 

3.  Take  the  temperature  next  morning  at  6  or  8  A.M.,  and  every  two 
hours  thereafter  until  6  or  8  P.M. 

Each  adult  animal  should  receive  2  c.c.  of  the  tuberculin  as  it  is  sent 
from  the  laboratory.  (The  tuberculin  sent  out  from  the  central  labora- 
tory at  Washington  is  already  diluted;  2.  c.c.  represents  0.25  c.c.  of  the 
concentrated  "  Old  Tuberculin  "  of  Koch.)  Yearlings  and  two-year-olds, 
according  to  size,  should  receive  from  1  to  1.5  c.c.  Bulls  and  very 
large  animals  may  receive  three  c.c.  The  injection  should  be  made 
beneath  the  skin  of  the  neck  or  shoulders  behind  the  scapula,  after 
washing  the  area  with  a  weak  carbolic  acid  solution. 

There  is  usually  no  marked  local  swelling  at  the  seat  of  the  injection. 

There  are  now  and  then  uneasiness,  trembling,  and  frequent  passage 
of  softened  dung.  There  may  also  be  slight  acceleration  of  the  pulse 
and  of  breathing. 

The  febrile  reaction  in  tuberculous  cattle  following  the  subcutaneous 
injection  of  tuberculin  begins  from  six  to  ten  hours  after  the  injection, 
reaches  the  maximum  nine  to  fifteen  hours  after  the  injection,  and 
returns  to  normal  eighteen  to  twenty-six  hours  after  the  injection. 

A  rise  of  two  or  more  degrees  Fahrenheit  above  the  maximum  tem- 
perature observed  on  the  previous  day  should  be  regarded  as  an  indica- 
tion of  tuberculosis.  For  any  rise  less  than  this  a  repetition  of  the  injec- 
tion after  four  or  six  weeks  is  highly  desirable. 

It  is  hardly  necessary  to  suggest  that  for  the  convenience  of  the  one 
making  the  test  the  animals  should  not  be  turned  out,  but  fed  and 
watered  in  the  stable.  It  is  desirable  to  make  note  of  the  time  of  feed- 
ing and  watering  and  of  any  temperature  fall  after  watering. 

The  tuberculin  should,  not  be  used  later  than  six  weeks  after  the 
date  on  the  bottle,  nor  if  there  is  a  decided  clouding  of  the  solution. 

Therapeutic  Uses  of  Tuberculin.— Tuberculin  was  first  used 
therapeutically,  by  Koch,63  shortly  after  its  discovery.  Hailed  with 
the  most  optimistic  enthusiasm,  its  possibilities  were  overestimated  and 
hopeless  cases  were  treated  unskillfully,  with  unsuitable  dosage.  The 
consequence  was  that  harm  was  done,  the  method  was  attacked  by 
Virchow  and  others  and  the  new  therapy  fell  into  almost  complete 
neglect.  At  present,  the  use  of  tuberculin  has  again  been  revived,  but 
with  greater  caution  and  with  a  thorough  understanding  of  its  limitations. 
The  tendency  has  been  toward  smaller  dosage  and  the  limitation  of  the 


63  Koch,  Deut.  med.  Woch.,  iii,  1891. 


THE  TUBERCLE  BACILLUS  611 

agent  to  early  cases.  No  two  institutions  use  tuberculin  in  exactly  the 
same  manner,  and  it  is,  therefore,  impossible  to  do  more  than  outline  the 
general  scheme  of  treatment.  It  must  never  be  forgotten,  however, 
that  all  forms  of  tuberculin  treatment  consist  in  an  "  active  immuniza- 
tion "  in  which,  for  the  time  being,  the  toxemia  of  the  patient  is  increased 
rather  than  neutralized.  It  is  obvious,  therefore,  that  only  cases  in 
which  the  process  is  not  a  very  acute  one,  are  at  all  suitable  for  treat- 
ment. The  general  principle  of  modern  tuberculin  therapy  seems  to  lie 
in  choosing  doses  so  small  that  no  marked  general  reaction  shall  follow. 
The  preparations  most  frequently  employed  are  Koch's  "Alttuber- 
culin,"  his  "TR,"  his  "Neu  Tuberkulin-Bazillen  Emulsion/'  and  the 
Bouillon  filtre  of  Denys.  Initial  doses  of  Alttuberculin  range  from  0.1 
to  0.01  of  a  milligram.  In  case  of  complete  absence  of  a  reaction, 
the  injection  may  be  repeated,  gradually  increasing,  about  twice  a  week. 
The  occurrence  of  a  reaction  should  be  the  signal  for  a  longer  interval 
and  a  slower  advance  in  the  size  of  the  dose. 

The  initial  dose  of  "TR"  is,  as  advised  by  Koch,64  about  0.002  mgm. 
This  usually  causes  no  reaction.  The  dose  is  doubled,  at  reasonable 
intervals,  up  to  1  mgm.  After  this,  further  increase  is  carefully  gauged 
by  the  clinical  indications.  The  maximum  dose  is  about  20  mgm. 

"Neu  Tuberkulin-Bazillen  Emulsion,"65  is  begun  with  a  dose  of 
0.001  mgm.  Gradual  increase  as  with  the  other  preparations  is  then 
practiced.  The  maximum  dose  is  about  10  mgm. 

Bouillon  filtre  has  been  used  chiefly  by  Denys 66  who  claims  ex- 
cellent results.  Denys  is  very  emphatic  in  advising  the  absolute 
avoidance  of  any  reaction.  He  begins  with  a  millionth  or  even  the 
tenth  of  a  millionth  of  a  cubic  centimeter  of  the  bouillon  and  increases 
with  extreme  caution.  His  dilutions  are  made  with  glycerin  broth. 

Active  immunization  with  tubercle  bacilli  of  reduced  virulence  has 
been  suggested  and  attempted  at  various  times  but,  so  far,  without 
definite  success.  No  strikingly  favorable  results  can  be  justly  claimed 
for  any  of  these  preparations. 

Passive  Immunization  in  Tuberculosis. — Numerous  attempts  have  been 
made  to  immunize  tuberculous  subjects  with  the  sera  of  actively  immune 


64  Koch,  Deut.  med.  Woch.,  xiv,  1897. 

83  Bandelier  and  Eoeplce,  "Lehrb.  d.  specifisch,  Tub.  Ther,/7  Wurzburg,  1908; 
Koch,  Deut.  med.  Woch.,  1901. 

88  Denys,  ' '  Le  Bouillon  filtre, ' '  Louvain,  1905. 


612  PATHOGENIC   MICROORGANISMS 

animals.  The  most  widely  used  method  of  producing  such  serum  is  that  of 
Maragliano. 

Maragliano's  Serum.Q7 — Maragliano  believes  that  a  toxalbumin  is  present 
in  tubercle-bacillus  cultures  which  is  destroyed  by  the  heating  employed  in 
the  usual  tuberculin  production.  He  procures  this  substance  by  nitration 
of  unheated  cultures  and  precipitation  with  alcohol  (tossina  prageipitata). 
He  furthermore  makes  an  aqueous  extract  of  the  bacillary  bodies.  With  these 
two  substances  he  immunizes  horses.  He  draws  blood  from  these  after  four 
to  six  months  of  treatment.  The  serum  is  extensively  used  in  Italy.  Its 
value  is,  at  present,  very  doubtful. 

Marmorek's  Serum.68 — Marmorek  claims  that  the  poisons  produced  by 
Bacillus  tuberculosis  depend  largely  upon  the  medium  on  which  it  is  grown. 
He  advanced  the  view  in  1903  that  the  substances  obtained  in  tuberculin 
were  not  the  true  toxins  of  the  tubercle  bacillus,  that  there  was  a  marked 
difference  between  these  and  the  poisons  elaborated  by  a  younger  (primitive) 
phase  of  the  bacillus  as  it  occurs  only  within  the  animal  body  or  on  media 
composed  of  animal  tissue.  He  consequently  grows  his  cultures  on  a  medium 
composed  of  a  leucotoxic  serum  (produced  by  inoculating  calves  with  guinea- 
pig  leucocytes)  and  liver  tissue.  Such  cultures,  he  claims,  contain  no  tuber- 
culin. To  the  sera  produced  by  immunization  with  these  cultures  he  attributes 
high  curative  powers. 


We  may  say  with  considerable  confidence  at  the  present  time  that 
no  method  of  passive  immunization  in  tuberculosis  has,  up  to  the  pres- 
ent, had  any  degree  of  success. 

Bacilli  Closely  Related  to  the  Tuberculin  Bacillus. — The  Bacillus  of 
Bovine  Tuberculosis. — Tuberculosis  of  cattle  (Perlsucht)  was  studied 
by  Koch  69  in  connection  with  his  early  work  on  human  tuberculosis. 
Koch 'did  not  fail  to  recognize  differences  between  the  reactions  to 
infection  in  the  bovine  type  of  the  disease  and  that  of  man.  He  attrib- 
uted these,  however,  to  the  nature  of  the  infected  subject  rather  than 
to  any  differences  in  the  infecting  agents.  This  point  of  view  met 
with  little  authoritative  contradiction,  until  Theobald  Smith,70  in  1898, 
made  a  systematic  comparative  study  of  bacilli  isolated  from  man  and 


6T  Maragliano,  Eerl.  klin.  Woch.,   1899;   Soc.  de  biol.,  1897. 

68  MarmoreJc,  Berl.  klin.    Woch.,   1903;   p.    1108;    Med.   Klinik,   1906. 

69  Koch,  Arb.  a.  d.  kais.  Gesimdheitsamt,  11,  1882. 

70  Th.  Smith,  Jour.  Exp.  Med.,  Ill,  1898. 


THE  TUBERCLE   BACILLUS  613 

from  cattle  and  pointed  out  differences  between  the  two  types.  The 
opinion  of  Smith  was  fully  accepted  by  Koch71  in  1901. 

Since  that  time,  the  question,  because  of  its  great  importance  to 
prophylaxis,  has  been  the  subject  of  many  investigations,  most  of  them 
confirming  Smith's  original  work.  Morphologically,  Smith 72  found 
that  the  bovine  bacilli  were  usually  shorter  than  those  of  the  human  type 
and  grew  less  luxuriantly  than  these  upon  artificial  media.  He  deter- 
mined, furthermore,  that,  grown  upon  slightly  acid  glycerin  bouillon, 
the  bovine  bacillus  gradually  reduces  the  acidity  of  the  culture  medium 
until  the  reaction  reaches  neutrality  or  even  slight  alkalinity.  Fluc- 
tuations after  this  do  not  exceed  0.1  to  0.2  per  cent  on  either  side  of 
neutrality.  In  the  case  of  the  human  bacillus,  on  the  other  hand,  there 
is  but  slight  reduction  of  the  acidity  during  the  first  weeks  of  growth; 
after  this  acidity  increases  and,  though  subject  to  fluctuations,  never 
reaches  neutrality.  This  behavior  is  probably  due  to  action  exerted 
upon  the  glycerin,  since  on  ordinary  bouillon  no  such  differences  between 
the  two  varieties  can  be  noticed.  These  observations  of  Smith  were 
confirmed  by  Ravenel,73  Vagedes,74  and  others. 

The  cultural  differences  between  the  two  types  have  been  studied 
with  especial  care  by  Wolbach  and  Ernst,75  and  Kossel,  Weber,  and 
Heuss.76  All  of  these  observers  bear  out  Smith's  contention  that 
luxuriance  and  speed  of  growth  are  much  more  marked  in  the  human 
than  in  the  bovine  variety.  Marked  differences,  furthermore,  have  been 
shown  to  exist  in  the  pathogenic  properties  of  these  bacilli  toward 
various  animal  species. 

Guinea-pigs  inoculated  with  the  bovine  type77  die  more  quickly  and 
show  more  extensive  lesions  than  those  infected  with  human  bacilli. 
The  difference  in  the  pathogenicity  of  the  two  organisms  for  rabbits  is 
sufficiently  striking  to  be  of  diagnostic  value.  The  bovine  bacilli  usually 
kill  a  rabbit  within  two  to  five  weeks;  the  human  bacilli  produce  a  mild 
and  slow  disease,  lasting  often  for  six  months,  and  occasionally  fail  to 
kill  the  rabbits  at  all. 

The  practical  importance  of  distinguishing  between  the  two  types, 


iiKoch,  Dent.  med.  Woch.,  1901. 

72T/i.  Smith,  Jour.  Exp.  Med.,  1905. 

73  Ravenel,  Lancet,  1901;  Univ.  Penn.  Med.  Bull.,  192. 

™Vagedes,  Zeit.  f.  Hyg.,  1898. 

75  Wolbach  and  Ernst,  ' '  Studies  from  the  Eockef eller  Inst., ' '  11,  1904. 

76  Kossel,  Weber,  und  Heuss,  Arb.  a.  d.  kais.  Gesundheitsamt,  1904  and  1905. 
n  Smith,  loc.  cit.,  and  Medical  News,   1902. 


614  PATHOGENIC   MICROORGANISMS 

of  course,  attaches  to  the  question  as  to  whether  the  bovine  and  the 
human  disease  are  mutually  intercommunicable.  This  has  been  dis- 
cussed in  the  preceding  section  dealing  with  the  human  type. 

Summary  of  the  Differentiation  between  Bovine  and  Human  Tubercle 
Bacilli. — Morphologically  the  bovine  bacillus  is  a  little  plumper  and 
thicker  than  the  human  type,  but  this  cannot  be  regarded  as  suf- 
ficiently constant  to  be  reliable  for  differentiation.  On  glycerin  broth, 
the  final  reaction  in  the  case  of  human  bacilli  is  considerably  acid,  the 
final  reaction  with  the  bovine  is  very  slightly  above  the  neutral 
point.  The  bovine  bacillus  does  not  grow  as  readily  as  the  human 
and  is  not  aided  by  the  addition  of  glycerin  to  the  media  to  the  same 
extent  as  the  human.  The  growth  of  the  human  bacillus  is  apt  to  be 
more  luxuriant  than  that  of  the  bovine,  especially  in  earlier  generations. 

As  to  virulence,  the  bovine  is  much  more  virulent  for  all  the  ordinary 
laboratory  animals  than  is  the  human.  The  difference  is  particularly 
marked  in  rabbits.  Small  doses  of  human  bacilli  inoculated  into  rab- 
bits will  kill  them  very  late,  and  if  quantities  of  less  than  0.1  of  a  milli- 
gram are  used  intravenously,  the  rabbits  may  live  for  longer  than  two 
months,  or  may  survive.  Similar  injection  of  the  bovine  type  into 
rabbits  kills  with  greater  regularity  and  more  extensive  lesions,  usually 
within  two  months. 

The  Bacillus  rf  Avian  Tuberculosis. — A  disease  resembling  in  many 
features  the  tuberculosis  of  man  is  not  uncommon  among  chickens, 
pigeons,  and  some  other  birds.  Koch  was  the  first  to  discover  in  the 
lesions  of  diseased  fowl,  bacilli  much  resembling  Bacillus  tuberculosis. 
It  was  soon  shown,  however,  by  the  studies  of  Nocard  and  Roux,78 
Mafucci,79  and  others,  that  the  bacillus  of  the  avian  disease  represented 
a  definitely  differentiate  species. 

Morphologically,  and  in  staining  characteristics,  the  bacillus  is 
almost  identical  with  that  of  the  human  disease.  In  culture,  however, 
growth  is  more  rapid  and  takes  place  at  a  temperature  of  41°  to  45°  C.80 
(the  normal  temperature  of  birds),  while  the  human  type  is  unable  to 
thrive  at  a  temperature  above  40°. 

The  organisms  grow  more  easily  than  do  either  the  human  or  bovine 
bacilli.  Colonies  appear,  on  glycerin  agar  within  a  week  and  cultiva- 
tion may  also  be  successful  on  media  without  glycerin.  It  is  char- 


ts Nocard  et  Roux,  Ann.  <le  1'inst.  Pasteur,  1887. 
™ Mafucci,  eit.  f.  Kyg.,  xi. 
80  Mafucci,  loc.  cit. 


THE  TUBERCLE  BACILLUS  615 

acteristic  of  the  avian  type  that  cultures  on  liquid  media  (glycerin 
broth)  grow  as  readily  within  the  liquid  as  on  the  surface  and  may 
even  become  homogeneous. 

Guinea-pigs,  very  susceptible  to  human  tuberculosis,  are  very 
refractory  to  infection  with  the  avian  type;  while,  on  the  other  hand, 
rabbits  which  are  resistant  to  the  human  type,  succumb  rapidly  to 
infection  with  avian  tuberculosis.81  Prolonged  cultivation  and  passage 
through  the  mammalian  body  is  said  to  cause  these  bacilli  to  approach 
more  or  less  closely  to  the  mammalian  type.  Conversely,  Nocard82 
claims  to  have  succeeded  in  rendering  mammalian  tubercle  bacilli 
pathogenic  for  fowl  by  keeping  them  in  the  peritoneal  cavities  of  hens 
in  celloidin  sacs  for  six  months. 

Recently  Koch  and  Rabinovitsch83  have  isolated  from  the  spleen  of 
a  young  man  dead  of  tuberculosis,  a  microorganism  which,  culturally, 
morphologically,  and  in  its  pathogenic  action  upon  birds,  seemed  to 
belong  to  the  avian  type.  Lowenstein84  describes  a  similar  organism 
cultivated  from  a  human  case  which  seems  to  be  a  transitional  type. 
Observations  of  this  order  ar^,  however,  too  few  at  the  present  time  to 
be  used  as  the  basis  of  a  definite  opinion  as  to  the  relationship  between 
the  two  varieties. 

Tuberculosis  in  Cold-blooded  Animals. — The  bacillus  isolated  by 
Dubarre  and  Terre85  resembles  Bacillus  tuberculosis  in  morphology  and 
in  a  certain  degree  of  acid-fastness.  It  grows  at  low  temperatures, 
15°  to  30°  C.  It  is  non-pathogenic  for  animals,  but  kills  frogs  within  a 
month.  Except  for  the  acid-fastness  it  has  little  in  common  with 
Bacillus  tuberculosis. 

Similar  acid-fast  bacilli  have  been  isolated  from  other  cold-blooded 
animals  (carp,  frogs,  turtles,  snakes)  by  many  observers. 

There  have  been  many  attempts  to  show  a  close  relationship  between 
the  tubercle  bacilli  of  cold-blooded  and  those  of  warm-blooded  animals. 
Moeller,  Hansemann,  Friedmann,  Weber,  Ktister,  and  others  have 
given  this  subject  particular  attention  and  it  has  gained  especial  interest 
because  of  the  recent  notorious  claims  of  Friedmann  that  he  had  suc- 
ceeded in  obtaining,  from  turtles,  a  strain  of  acid-fast  bacilli  which  could 


et   Gamalcw,   Arch.  do  nu''d.  expor.,   1891;    Courmont  et  Dor,  Arch,  de 
mod.  oxp.,   1S91. 

l,  Ann.  de  1'inst.  Pasteur,   1S9X. 

mid  Ktiltiiiovitxi'h.  Viirh.  Arch.,  Beihoft  to  Bd.  190,  1907. 
n*Lowensteinf  quoted   from    Koch   and  Kabinovitsch,  loe.  cit. 
MDi/tbarire  et  Tcrre,  Conipt.  rend,  de  la  soc.  de  biol.,  1897. 


61(5  PATHOGENIC   MICROORGANISMS 

be  successfully  used  in  actively  immunizing  human  beings.  In  1903 
Friedmann86  described  two  cases  of  spontaneous  infection  of  a  salt-water 
turtle  (Chelone  corticata)  with  acid-fast  bacilli,  presenting  lesions  in 
the  lungs  which  simulated  pulmonary  tuberculosis  in  the  human  being 
(cavity  formation  and  miliary  nodules) .  The  organisms  cultivated  from 
these  lesions  presented  much  similarity  to  those  of  the  human  type  and, 
according  to  Friedmann,87  unlike  other  acid-fast  bacilli  of  cold-blooded 
animals,  could  be  grown  at  37.5°  C.  As  a  possible  human  origin  for  the 
turtle  infections  Friedmann  mentions  that  the  attendant  who  fed  these 
turtles  suffered  from  a  double  pulmonary  tuberculosis. 

Upon  inoculation  into  guinea-pigs  localized  lesions  only  were  pro- 
duced, and  dogs,  rats,  and  birds  were  immune.  The  implication  of 
Friedmann's  work  is  that  his  culture  represented  a  human  strain  attenu- 
ated for  man  by  passage  through  the  turtle,  although,  as  far  as  we  are 
aware,  no  definite  statement  as  to  this  was  made. 

Summarizing  the  work  of  many  investigators  (Weber,  Taute, 
Kiister,  Allegri,  Bertarelli,  and  others)  Kiister  88  makes  a  statement 
which  is,  in  essence,  as  follows:  In  the  carp,  in  snakes,  turtles,  and  frogs 
spontaneous  "  tuberculosis  "  may  occur.  The  organisms  which  cause 
these  diseases  are  specific  for  cold-blooded  animals,  similar  in  many  re- 
spects to  the  tubercle  bacillus  of  warm-blooded  animals,  but  in  the  latter 
do  not  produce  progressive  disease.  Human,  bovine,  and  avian  tubercle 
bacilli  inoculated  into  cold-blooded  animals  can  produce  lesions  which 
histologically  simulate  tuberculosis.  These  microorganisms  can  remain 
a  year  in  cold-blooded  animals  without  losing  their  pathogenicity  for 
guinea-pigs.  Mutation  of  the  tubercle  bacillus  of  warm-blooded  animals 
into  cold-blooded  ones  has  not  been  proven. 

P'or  these  reasons  it  is  quite  impossible  to  exclude,  in  the  apparently 
positive  work  of  Friedmann  and  others,  the  isolation  of  a  true  "  cold- 
blooded" type  organism,  rather  than  a  mutation  form  originally  of 
the  warm-blooded  type.  What  Friedmann's  present  claims  in  this 
respect  are  for  his  culture  has  not  been  stated  as  far  as  we  know.  The 
possibility  of  a  positive  immunizing  value  of  organisms  isolated  from 
cold-blooded  animals  in  human  beings,  though  remote,  is  not  out  of 
question.  The  problem  is  so  serious  and  important,  and  the  experience 


**  Friedmann,  D.  Metl.  Woch.,  No.  2,  Jan.,  1903,  25. 

^Friedmann.  D.  Med.   Woch.,  No.   26,  464,   1903,   and  Centralbl.  f.  Bakt.,   I, 
xxxiv,  1903,  also  Zeitschr.  f.  Tuberkulose,  iv,  Heft  5,  1903. 
**Kolle  und   Wassermann's  Handbuch,  2d  edition,  v,  767. 


THE  TUBERCLE   BACILLUS  617 

of  many  workers  is,  so  far,  so  inconclusive  that  the  time  has  not  come  for 
commercial  exploitation  and  the  cruel  deceptions  of  false  hopes.  The 
subject,  however,  deserves  carefully  controlled  further  investigations. 

Bacillus  of  Timothy. —  Moeller  isolated  from  timothy-grass  and  from 
the  dust  in  haylofts  acid-fast  bacilli,  like  Bacillus  tuberculosis.  They 
grow  rapidly  on  agar,  soon  showing  a  deep  red  or  dark  yellow  color. 

Bacillus  butyricus  (Butter  Bacillus). — Slightly  acid-fast  bacilli 
resembling  Bacillus  tuberculosis  have  been  isolated  from  milk  and  butter 
by  Petri,89  Rabinovitsch,90  Korn,91  and  others. 

These  bacilli  are  easily  differentiated  from  Bacillus  tuberculosis 
culturally.  They  are  slightly  pathogenic  for  guinea-pigs,  but  not  for 
man. 

Bacillus  smegmatis  and  the  bacillus  of  leprosy  will  be  discussed  in 
separate  sections.  The  differentiation  of  these  organisms  by  staining 
reactions  has  been  discussed  in  the  section  on  staining  methods. 

s9  Petri,  Arb.  a.  d.  kais.  Gesundheitsamt,  1897. 
MRabinovitsch,  Zeit.  f.  Hyg.,  1897. 
91  Korn,  Cent.  f.  Bakt.,  1899. 


CHAPTER  XXX 

THE    SMEGMA   BACILLUS   AND    THE    BACILLUS    OF    LEPEOSY 

BACILLUS    SMEGMATIS 

IN  1884,  Lustgarten  l  announced  that  he  had  succeeded  in  demon- 
strating, in  a  number  of  syphilitic  lesions,  a  characteristic  bacillus, 
which  he  declared  to  be  the  etiological  factor  in  the  disease.  The  great 
importance  of  the  subject  of  Lustgarten's  communication  caused 
numerous  investigators  to  take  up  the  study  of  the  microorganisms  found 
upon  the  genitals  of  normal  and  diseased  individuals.  As  a  result  of 
these  researches  the  presence  of  the  Lustgarten  bacilli  upon  the  genitals 
of  many  syphilitics  was  confirmed;  but  at  the  same  time  bacilli,  which 
in  all  essential  particulars  were  identical  with  them,  were  found  in  the 
secretions  about  the  genital  organs  and  anus  of  many  normal  persons. 
The  first  to  throw  doubt  upon  the  etiological  significance  of  Lustgarten's 
bacillus,  and  to  describe  in  detail  the  microorganism  now  recognized  as 
Bacillus  smegmatis,  were  Alvarez  and  Tavel.2  Similar  studies  were 
made  soon  afterward  by  Klemperer,3  Bitter,4  and  others. 

The  smegma  bacilli  are  now  known  to  occur  as  harmless  sapro- 
phytes in  the  preputial  secretions  of  the  male,  about  the  external  genital 
organs  of  the  female,  and  within  the  folds  of  thighs  and  .buttocks.  They 
are  usually  found,  in  these  situations,  in  clumps  upon  the  mucous  mem- 
brane, and  occasionally  in  the  superficial  layers  of  the  epithelium,  intra- 
and  extra-cellularly. 

Morphology. — The  smegma  bacilli  are  very  similar  to  tubercle 
bacilli,  but  show  greater,  variations  in  size  and  appearance  than  do  the 
latter.  In  length  the  individuals  may  vary  from  two  to  seven  micra. 
They  are  usually  straight  or  slightly  curved,  but  according  to  Alvarez 
and  Tavel  may  show  great  polymorphism,  including  short  comma-like 
forms,  and  occasional  S-shaped  spiral  forms. 

1  Lustgarten,  Wien.  med.  Woch.,  47,  1884. 

2  Alvarez  et  Tavel,  Arch.  d.  physiol.  norm,  et  path.,  Oct.,  1885. 
8  Klemperer,  Deut.  med.  Woch.,  xi,  1885. 

4  Bitter,  Virchow's  Arch.,  ciii. 

618 


BACILLUS  SMEGMATIS  619 

They  are  not  easily  stained,  and  though  less  resistant  in  this  respect 
than  the  tubercle  bacillus,  they  yet  belong  distinctly  to  the  group  of 
acid-fast  bacilli.  Once  stained  by  the  stronger  dyes,  such  as  carbol- 
fuchsin  or  anilin-water-gentian -violet,  they  are  tenacious  of  the  dye, 
though  less  so  than  tubercle  bacilli. 

The  identification  of  the  smegma  bacillus  by  staining  methods 
has  become  of  practical  importance  since  Fraenkel,5  Miiller,6  and 
others  have  demonstrated  the  occasional  presence  of  acid-fast  bacilli, 
probably  of  the  smegma  group,  in  sputum,  and  in  secretions  from  the 
tonsillar  crypts  and  throat.  The  methods  of  differentiation  which  have 
been  found  most  practical  are  those  which  depend  upon  differences  in 
the  retention  of  stain  shown  by  these  bacilli.  While  it  may  be  stated 
as  a  general  rule  that  the  smegma  bacilli  are  more  easily  decolorized 
than  tubercle  bacilli,  it  is  nevertheless  important  that  a  control,  as  sug- 
gested by  Wood,  be  made  with  known  tubercle  bacilli  whenever  a  slide 
of  suspected  smegma  bacilli  is  examined.  For  the  actual  differentiation 
an  excellent  method  is  that  of  Pappenheim,  described  in  detail  in  the 
section  on  Staining,  page  125.  This  method  depends  upon  the  fact 
that  prolonged  treatment  with  alcohol  and  rosolic  acid  decolorizes  the 
smegma  bacilli  but  not  the  tubercle  bacilli.  Coles7  has  stated  that 
smegma  bacilli  will  resist  Pappenheim's  decolorizing  agent  for  four  hours 
at  the  most,  while  tubercle  bacilli  will  retain  the  stain,  in  spite  of  such 
treatment,  for  as  long  as  twenty-four  hours. 

The  smegma  bacilli  have  no  pathogenic  significance.  They  are 
found  upon  human  beings  as  harmless  saprophytes,  and  all  attempts  to 
infect  animals  have  so  far  been  unsuccessful.  They  are  cultivated 
with  great  difficulty,  first  cultivations  from  man  being  successful  only 
upon  the  richer  media  containing  human  serum  or  hydrocele  fluid. 
After  prolonged  cultivation  upon  artificial  media  they  may  be  kept 
alive  upon  glucose  agar  or  ascitic  agar.  Their  growth  is  slow;  and  the 
colonies,  appearing  within  five  or  six  days  after  inoculation,  are  yellowish 
white,  corrugated,  and  not  unlike  tubercle-bacillus  colonies. 


5Fraenlcel,  Bcrl.  klin.  Woeh.,  1898. 
41  Miiller,  Deut.  med.  VVoch.,  1898. 
7  Coles,  Jour,  of  State  Med.,  1904. 


620  PATHOGENIC   MICROORGANISMS 


LEPROSY    BACILLUS    LEPR^E 

The  bacillus  of  leprosy  was  first  seen  and  interpreted  as  the 
etiological  factor  in  the  disease  in  1879,  by  G.  Arrnauer  Hansen, g 
a  Norwegian  observer.  Hansen  found  the  bacilli  in  the  tissues  of  the 
nodular  lesions  of  patients,  lying  in  small  clumps,  intra-  and  extra- 
cellularly,  as  well  as  in  the  serum  oozing  from  the  tissue  during  its 
removal.  Hansen's  observation  was  the  fruit  of  over  six  years  of  careful 
study  and  as  to  his  priority  in  making  this  discovery,  there  can  be 
no  doubt.  Almost  simultaneously  with  his  publication,  however, 
Neisser  9  published  similar  results,  obtained  by  him  during  a  brief  stay 
at  Bergen,  during  the  preceding  summer.  The  bacilli  described  by 
these  workers  are  now  recognized  as  being  unquestionably  the  cause  of 
the  various  forms  of  the  disease  known  as  leprosy. 

Morphology  and  Staining. — The  leprosy  bacillus  is  a  small  rod 
measuring  about  5  to  7 '/*  in  length  and  has  a  close  morphological 
resemblance  to  Bacillus  tuberculosis,  except  in  that  it  is  less  apt  to  dis- 
play the  beaded  appearance  and  is  slightly  less  slender  than  the  latter. 
It  is  non-motile,  possesses  no  flagella,  and  forms  no  spores. 

Like  tubercle  bacilli,  the  leprosjr  bacilli  belong  to  the  class  of 
so-called  acid-fast  bacteria,  being  stained  with  much  difficulty;  but 
when  once  stained  they  are  tenacious  of  the  color,  offering  consider- 
able resistance  to  the  decolorizing  action  of  acids.  It  is  necessary 
for  differential  diagnosis,  however,  to  note  that  both  the  difficulty  of 
staining  and  the  resistance  to  decolorization  are  less  marked  in  the  case 
of  this  microorganism  than  in  the  case  of  Bacillus  tuberculosis.  It  was 
this  peculiar  behavior  to  stains  that  caused  the  delay  of  several  years  in 
Hansen's  publications,  since  he  failed  in  obtaining  good  morphological 
specimens  until  the  work  of  Koch  upon  bacterial  staining  had  supplied 
him  with  proper  methods.  The  bacillus  is  stained  most  easily  with 
anilin-water-gentian-violet  or  with  carbol-fuchsin  solution.  Stained  by 
Gram's  method,  it  is  not  decolorized  and  appears  a  deep  blue.  Differ- 
ential staining  by  the  Ziehl-Neelson  method  shows  the  bacillus  stained 
red  unless  decolorization  by  means  of  the  acid  and  alcohol  are  prolonged 
for  an  unusual  time.  A  differentiation  from  tubercle  bacilli  by  virtue  of 
greater  ease  of  decolorization  is  of  value  only  in  the  hands  of  those 
having  much  experience  with  these  bacilli,  and  follows  no  regular  laws 

8  Hansen,  Virch.  Arch.,  79,  1879. 

9  Neisser,  Breslauer  arztl.  Zeitschr.,  20,  1879. 


LEPROSY  BACILLUS  LEPILE  621 

of  acid-strengths  or  time  of  application  which  can  be  generally  applied 
by  the  inexperienced.  In  tissues,  the  bacilli  are  easily  stained  by  the 
methods  used  for  staining  tubercle  bacilli.  The  sections  are  left  in  the 
Ziehl  carbol-fuchsin  solution  either  from  two  to  twelve  hours  at  incu- 
bator temperature  or  for  twenty-four  hours  at  room  temperature. 
Subsequent  treatment  is  that  employed  in  the  case  of  tuberculous  tissue 
sections  (see  p.  132). 

Cultivation. — Cultivation  of  the  leprosy  bacillus  has  not  met  with 
success.  Hansen  and  others  who  have  approached  the  problem  with  a 
thorough  knowledge  of  the  microorganism,  combined  with  a  competent 
bacteriological  training,  have  failed  in  all  their  attempts.  The  numer- 
ous positive  results  reported  by  observers  have  always  lacked  adequate 
confirmation.  Recently,  Host,10  of  the  British  Army  Medical  Corps, 
claimed  success  in  cultivation  of  leprosy  bacilli  upon  salt-free 
bouillon,  his  point  of  departure  being  the  previous  observation  that 
salt-free  media  favored  the  growth  of  tubercle  bacilli.  His  results  have 
not  been  confirmed. 

In  1909  Clegg  u  succeeded  in  growing  an  acid-fast  bacillus  from 
leprous  tissue,  obtaining  his  results  by  inoculating  leprous  material 
upon  agar  plates  upon  which  ameba  coli  had  been  grown  in  symbiosis 
with  other  bacteria.  On  such  plates  the  acid-fast  bacilli  multiplied, 
and  subsequently,  pure  cultures  were  obtained  by  heating  the  cultures 
to  60°  C.,  which  destroyed  the  ameba  colic  and  other  bacteria.  These 
results  were  confirmed  by  other  workers  and,  soon  after  that,  Duval 12 
not  only  succeeded  in  repeating  Clegg's  experiments,  but  obtained  cul- 
tures of  an  acid-fast  bacillus  directly  from  leprous  lesions  without  the 
aid  of  ameba.  He  first  observed  that  the  leprosy  organism  would  multi- 
ply around  a  transplanted  piece  of  leprous  tissue  upon  ordinary  blood 
agar  tubes  upon  which  influenza  bacilli  and  meningococci  were  grown. 
He  concluded  that  such  growth  depended  upon  chemical  changes  in 
the  media  and  believed  the  formation  of  amino-acids  essential  for  the 
initial  growth.  The  method  he  subsequently  described  depended  upon 
supplying  these  substances  either  by  adding  tryptophan  to  nutrient 
agar  or  by  pouring  egg  albumen  and  human  blood  serum  in  Petri  dishes, 
inspissating,  at  70°  C.,  for  three  hours  and,  after  inoculating  with 
leprous  tissue,  adding  a  1  per  cent  solution  of  trypsin.  Indirectly  the 
same  result  was  obtained  by  employing  culture  media  containing  albu- 

10Eost,  Brit.  Med.  Jour.  1,  1905. 

**  Clegg,  Philippine  Jour,  of  Se.,  iv,  1909. 

17  Duval,  Jour.  Exp.  Med.,  xii,  1910,  and  ibid.,  15,  1912. 


622  PATHOGENIC   MICROORGANISMS 

minous  substances  and  inoculating  with  bacteria  capable  of  producing 
amino-acids  from  the  medium.  After  leprosy  bacilli  had  been  grown 
on  for  several  generations,  they  could  easily  be  cultivated  on  agar  slants 
without  special  additions  or  preliminary  treatment. 

In  spite  of  extensive  work  upon  this  very  important  problem  opin- 
ions are  still  divided  as  to  the  specific  nature  of  the  organisms  cul- 
tivated by  Clegg  and  by  Duval.  Animal  experiments  with  these  cul- 
tures have  remained  inconclusive.  The  cultures  after  prolonged  preser- 
vation upon  artificial  media  grow  heavily,  often  lose  their  acid-fast 
characteristics,  develop  into  strep tothrix-like  or  diphtheroid  forms  and 
become  markedly  chromogenic,  all  these  characteristics  suggesting 
saprophytism. 

In  a  recent  communication,  Duval13  states  his  opinion  as  follows: 
From  29  cases  of  leprosy,  22  successive  cultivations  of  acid-fast  bacilli 
were  made;  in  14  of  them  a  chromogenic  organism,  similar  to  that  of 
Clegg,  was  found.  This  grows  either  as  a  non-acid-fast  streptothrix  in 
subsequent  cultivations  or  as  non-acid-fast  diphtheroid  forms.  From 
eight  cases  an  organism  distinctly  different  from  the  former  was  cul- 
tivated which  grows  only  on  specific  media  and  by  serological  tests 
seems  to  give  reaction  which  differentiates  it  from  Clegg's  organism. 
Duval  believes  that  there  is  no  reason  to  assume  specific  etiological 
relationship  for  the  first  organism  mentioned.  In  the  case  of  the  second, 
he  admits  that  not  sufficient  proof  has  been  brought,  but  states  his  belief 
that  its  etiological  significance  is  probable. 

Pathogenicity. — Numerous  attempts  to  transmit  leprosy  to  ani- 
mals by  inoculation  have  been  unsuccessful.  Nicolle,14  however,  has 
recently  claimed  successful  experiments  upon  monkeys  (macacus)  in 
whom  inoculation  with  tissue  from  :nfected  human  beings  was  followed, 
in  sixty-two  days,  by  the  development  of  a  small  nodule  at  the  site  of 
inoculation,  in  which,  upon  excision,  leprosy  bacilli  were  found.  In 
most  cases,  however,  inoculation  has  given  rise  merely  to  a  transient 
inflammatory  reaction. 

Among  human  beings,  leprosy  has  been  a  widely  spread  disease  since 
the  beginning  of  history,  and  much  evidence  is  found  in  ancient  litera- 
ture which  testifies  to  a  wide  distribution  of  the  disease  long  before  the 
Christian  era  and  throughout  the  Middle  Ages.  At  the  present  day, 
leprosy  is  most  common  in  the  eastern  countries,  especially  in  India  and 


13  Duval,  Jour,  of  Inf.  Dis.,  xi,  1912. 
"Nicolle,  Sem.  medieale,  10,  1905. 


LEPROSY   BACILLUS  LEPR^  623 

China.  In  Europe  the  disease  is  found  in  Norway,  in  Russia,  and  in 
Iceland.  In  other  European  countries,  while  the  disease  occurs,  it  is 
not  at  all  common.  In  the  United  States,  there  are,  according  to  Osier, 
three  important  centers  of  leprosy  situated  in  Louisiana,  in  California, 
and  among  the  Norwegian  settlers  in  Minnesota.  The  disease  is  also 
present  in  several  provinces  of  Canada.  In  all  countries  in  which 
segregation  of  lepers  is  rigidly  practiced,  the  disease  is  diminishing.  In 
Norway,  according  to  Hansen,  proper  sanitary  measures  have  reduced 
the  number  of  lepers  from  2870  in  1856,  to  577  in  1900. 

Clinically,  the  disease  appears  in  two  chief  varieties,  tubercular 
leprosy  and  the  so-called  anesthetic  leprosy.  In  the  former  variety, 
hard  nodular  swellings  appear,  usually  in  the  face,  but  often  on  other 
parts  of  the  body  as  well.  These  lead  to  frightful  disfigurement  and 
are  accompanied  by  a  falling-out  of  hair  and  a  loss  of  sensation  in  the 
affected  areas.  In  the  anesthetic  form,  there  is  usually  at  first  pain  in 
definite  areas  of  the  extremities  and  the  trunk,  which  is  soon  followed 
by  the  formation  of  flat  or  slightly  raised  pigmented  areas,  within  which 
there  is  absolute  anesthesia  with,  later,  atrophy  and  often  secondary 
necrosis  in  the  atrophied  parts.  The  disease  is  usually  chronic  in  its 
course. 

The  bacilli  are  found  in  large  numbers  in  the  cutaneous  lesions.  In 
the  knobs  of  the  nodular  variety,  they  lie  in  clumps  between  the  con- 
nective-tissue cells  and  within  the  large  spheroidal  cells  which  make  up 
the  nodules.  They  are  found,  also,  in  advanced  cases,  in  the  liver  and 
in  the  spleen,  lying  within  the  cells,  and,  to  a  slighter  extent,  in  the 
intercellular  spaces.  They  have  also  been  found  within  the  kidneys, 
the  endothelium  of  the  blood-vessels,  and  in  the  testicles.15  In  the  blood, 
the  bacilli  have  frequently  been  demonstrated,  especially  during  the 
febrile  attacks  which  occur  during  the  disease.  Westphal  and  Uhlen- 
hut16  have  found  the  bacilli  within  the  central  nervous  system,  and 
these  observers,  as  well  as  others,  have  found  them  lying  within  the 
substance  of  the  peripheral  nerves,  thus  explaining  the  anesthesia.  A 
fact  of  importance  to  the  question  of  transmission  is  the  observation 
made  by  various  observers,  more  especially  by  Sticker,  that  the  bacilli 
are  found  with  great  regularity  and  in  considerable  numbers  in  the  nasal 
secretions  of  persons  suffering  from  the  disease.  Sticker  is  inclined  to 
regard  the  nose  as  the  primary  path  of  infection.  Whether  or  not  this 


a  Slicker,  Miinch.  mod.  Woch.,  39,  1897. 

16  Westphal  imd  UMenhut,  Klin.  Jahrb.,  1901. 


624  PATHOGENIC   MICROORGANISMS 

be  true  can  not,  at  present,  be  decided.  As  a  source  of  infection,  how- 
ever, the  nasal  mucus  and,  secondarily,  the  saliva,  are  certainly  the 
vehicles  by  which  large  numbers  of  the  bacilli  leave  the  infected  patient, 
and  therefore,  tend  to  spread  the  disease. 

The  contagiousness  of  leprosy  is  far  less  than  is  that  of  most  other 
bacterial  diseases.  Physicians  and  others  who  come  into  direct  contact 
with  large  numbers  of  leprous  patients,  the  ordinary  precautions  of 
cleanliness,  rarely  contract  the  disease.  On  the  other  hand,  intimate 
contact  with  lepers  without  such  precautions  is  the  only  possible 
means  of  transmission.  The  demonstration  of  leprosy  bacilli  in 
dust,  soil,  etc.,  must  always  be  looked  upon  with  suspicion,  since, 
apart  from  actual  human  inoculation,  there  is  no  method  of  positively 
differentiating  the  bacilli  from  similar  acid-fast  organisms.  Instances 
of  transmission  by  contact  are  on  record,  not  the  least  famous  of 
which  is  the  case  of  Father  Damien,  who  contracted  the  disease  while 
taking  care  of  the  lepers  upon  the  island  of  Molokai.  Hansen  states 
that  in  his  knowledge  no  case  of  leprosy  can  be  found  in  which  careful 
examination  of  the  past  history  will  not  reveal  direct  contact  with  a 
previous  case.  Direct  inoculation  of  the  human  being  with  material 
from  a  leprous  patient  has  been  successfully  carried  out  by  Arning,17 
upon  a  Hawaiian  criminal.  In  this  case  a  piece  of  a  leprous  nodule 
was  planted  into  the  subcutaneous  tissue  of  the  left  arm.  One  month 
after  the  inoculation,  pain  appeared  in  the  arm  and  shoulder,  and  four 
and  a  half  months  later  a  typical  leprosy  nodule  was  formed.  Four 
years  after  the  inoculation,  the  patient  was  a  typical  leper. 

Although  our  inability  to  cultivate  the  leprosy  bacillus,  and  the  lack 
of  success  attending  animal  inoculation,  have  made  it  impossible  to 
study  more  closely  the  toxic  action  of  this  microorganism,  there  is, 
nevertheless,  some  evidence  which  points  toward  the  production  of  a 
poisonous  substance  of  some  kind  by  the  bacillus.  Rost,18  who  claims 
to  have  cultivated  the  bacillus,  manufactured  from  his  cultures,  by  the 
technique  for  the  production  of  "Old  Tuberculin,"  a  substance  which  he 
called  "leprolin,"  and  which  he  employed  therapeutically  in  the  same 
manner  in  which  tuberculin  is  employed  in  tuberculosis.  As  stated  be- 
fore the  results  of  Rost  still  lack  confirmation. 

Of  far  greater  importance,  both  in  demonstrating  the  probability 
of  the  existence  of  a  definite  toxin  as  well  as  in  indicating  the  close 


17  Arning,  Vers.  <1.  Naturfor.  u.  Aerzte,  1886. 

18  Kost,  loc.  cit. 


RAT  LEPROSY  625 

relationship  between  the  leprosy  bacillus  and  the  Bacillus  tuberculosis, 
are  the  investigations  upon  the  action  of  tuberculin  upon  leprous 
patients.  When  tuberculin  is  administered  to  lepers,  a  febrile  reac- 
tion occurs  usually  twenty-four  or  more  hours  after  the  adminis- 
tration. The  fever  differs  from  that  produced  by  the  use  of  the 
same  substance  in  tuberculous  patients  in  that  it  is  of  late  occurrence 
and  lasts  considerably  longer.  At  the  same  time,  there  may  be 
marked  redness  and  tenderness  of  the  nodules.  In  isolated  cases, 
Babes  19  has  noticed  alarmingly  high  and  prolonged  fever  together 
with  systemic  symptoms  such  as  nausea,  headache,  and  even  uncon- 
sciousness, following  the  injection  of  tuberculin.  The  same  writer 
claims  to  have  extracted  from  the  organs  of  lepers,  which  contained 
enormous  numbers  of  bacilli,  substances  which  showed  an  action  similar 
to  that  of  the  tuberculin. 

Until  recently  all  therapeutic  methods  applied  to  leprosy  have  been 
discouraging.  In  1914  Heiser  reported  on  the  treatment  of  12  cases 
by  intramuscular  injections  of  Chaulmoogra  oil.  McDonald  and  Dean, 
Sir  Leonard  Rogers  and  others  have  used  this  oil  and  its  derivatives 
since  then  with  encouraging  results.  McDonald  and  Dean  report  186 
cases  treated  from  1918  to  1919,  25  of  whom  were  discharged  as  "  clin- 
ically and  bacteriologically "  free  during  this  period.  They  used 
intramuscular  injections  of  ethyl  esters  of  the  fatty  acids  of  the  Chaul- 
moogra oil.  This  form  of  treatment  is  being  further  studied  by  them 
and  many  others.  For  details  we  refer  the  reader  to  their  communica- 
tion in  the  U.  S.  Public  Health  Reports,  August  20,  1920,  Vol.  35. 
No.  34, 

RAT    LEPROSY 

Stefansky20  first  observed  this  disease  among  rats  in  Odessa,  and 
since  then  it  has  been  observed  in  Berlin  (Rabinovitsch  21),  in  London 
(Dean22),  in  New  South  Wales  (Tidswell 23) ,  and  in  San  Francisco 
(Wherry  24  and  McCoy  25) .  The  disease  occurs  spontaneously  among 

19 Babes,  in  Kolle  und  Wasscrmann,  "Handbuch,"  etc.,  Erst.  Ergiinz.  Bd.,  1907. 

20  Stefansky,  Centralbl.  f.  Bakt.,  xxxiii,  481. 

21  EabinovUsch,  Centralbl.  f .  Bakt.,  xxxiii,  577. 

22  Dean,  Centralbl.  f.  Bakt.,  xxxiv,  222;  Jour.  Hyg.,  xcix. 

23  TiUswell,   cited   by   Brinkerhoff   in    "The   Eat   and    Its   Relation   to   Publie 
Health/'  Treas.  Dept.,  Wash.,  1910. 

24  Wherry,  J.  A.  M.  A.,  June  6,  1908. 

25  McCoy,  Rep.  U.  S.  P.  H.  and  M.  H.  S.,  xxiii,  981. 


626  PATHOGENIC   MICROORGANISMS 

house  rats  and  is  characterized  by  subcutaneous  induration,  swelling  of 
lymph  nodes,  with,  later,  falling  out  of  the  hair,  emaciation,  and  some- 
times ulceration.  Its  course  is  protracted  and  rats  may  live  with  it 
for  six  months  or  a  year.  When  a  rat  suffering  from  this  disease  is  dis- 
sected there  is  usually  found,  under  the  skin  of  the  abdomen  or  flank,  a 
thickened  area  which  has  the  appearance  of  adipose  tissue  except  that 
it  is  more  nodular  and  gray  and  less  shiny  than  fat.  It  is  so  like  fat, 
however,  that  it  is  often  overlooked  by  those  unfamiliar  with  the 
condition.  In  this  area  acid-fast  bacilli  looking  like  the  Bacillus  leprae 
are  found  in  large  numbers.  These  bacilli  are  also  found  in  the  lymph 
nodes  and  sometimes  in  small  nodules  in  the  liver  and  lung. 

The  disease  can  be  transmitted  experimentally  from  rat  to  rat  and 
probably  is  transmitted  naturally  from  rat  to  rat  by  the  agency  of 
fleas  (Wherry,  McCoy).  Although  clinically  not  exactly  like  human 
leprosy  the  condition  is  sufficiently  like  it  to  arouse  much  hygienic 
interest.  The  distribution  of  the  disease  in  various  parts  of  the  world 
does  not  correspond  with  the  distribution  of  leprosy.  A  peculiar 
feature  of  its  distribution  is  the-  fact  that  in  San  Francisco,  as  the  writer 
was  told  by  McCoy,  almost  all  the  rats  that  suffered  from  this  disease 
came  from  the  district  in  which  the  retail  meat  business  is  located, 
known  as  "Butchertown."  The  organisms  were  made  to  multiply  in 
vitro  by  Zinsser  and  Gary  in  plasma  preparations  of  growing  rat  spleen. 
Chapin  has  succeeded  in  cultivating  them  by  a  method  analogous  to 
the  trypsin-egg  albumen  method  employed  by  Duval.  In  the  experi- 
ments of  Zinsser  and  Gary  it  was  found  that  although  the  organisms 
may  retain  their  acid-fast  characteristics  for  many  weeks  within  leu- 
cocytes they  degenerate  rapidly  within  the  spleen  cells,  a  fact  which 
seems  to  have  some  bearing  on  the  mechanism  of  resistance  possessed 
by  the  body  against  acid-fast  organisms, 


CHAPTER  XXXI 

BACILLI  OF  THE  COLON-TYPHOID-DYSENTERY  GROUP 

THE    COLON    BACILLI 

THE  bacilli  belonging  to  this  group  of  microorganisms,  while  present- 
ing great  differences  in  their  pathogenic  characteristics,  possess  many 
points  of  morphological  and  biological  similarity  which  have  made  their 
differentiation  extremely  difficult.  Among  pathogenic  bacilli,  they  are 
probably  the  ones  most  commonly  encountered  and  because  of  the  fact 
that  some  of  them  are  specifically  pathogenic,  while  others  are  essen- 
tially saprophytic  and  are  pathogenic  only  under  exceptional  conditions, 
the  necessity  of  accurate  differentiation  is  a  daily  occurrence  in  bacteri- 
ological laboratories.  It  has  been  through  the  study  of  this  group  par- 
ticularly that  many  of  the  modern  differential  methods  of  bacteriology 
have  been  developed. 

The  group  includes  the  colon  bacillus  and  its  allies,  the  typhoid 
bacillus,  the  paratyphoid  organisms,  the  several  varieties  of  dysentery 
bacillus  and  numerous  closely  related  species,  and  Bacillus  fecalis  alka- 
ligenes.  Closely  related  to  the  group  though  not  properly  within  it, 
are  Bacillus  lactis  aerogenes,  B.  acidi  lactici,  bacilli  of  the  Friedlander  or 
mucosus  capsulatus  group,  and  a  number  of  less  important  subdivisions 
of  this  last  group. 

All  bacilli  of  the  group  possess  morphological  characteristics  which, 
although  exhibiting  slight  differences,  are  insufficient  to  permit  accurate 
morphological  diagnosis.  They  are  none  of  them  spore-bearing.  Stained 
by  Gram's  method  they  are  decolorized. 

Cultivated  upon  artificial  media,  they  grow  readily  both  at  room  and 
at  incubator  temperatures.  None  of  them  liquefies  gelatin.  Though 
showing,  often,  distinct  differences  in  the  speed  and  luxuriance  of  growth 
upon  ordinary  media,  these  differences  are,  nevertheless,  too  slight  to 
become  the  basis  of  differentiation. 

In  order  to  distinguish  between  the  individual  members  of  this 
group,  therefore,  we  are  forced  to  a  careful  biological  and  cultural 
study.  This  is  carried  out  by  the  observation  of  the  cultural  character- 

627 


628  PATHOGENIC  MICROORGANISMS 

istics  upon  special  media  and  by  the-  study  of  serum  reactions  in  specific 
immune  sera.  Our  mainstays  in  the  accurate  differentiation  of  these 
bacilli  are  their  fermentative  actions  upon  carbohydrate  media,  and 
their  agglutinating  reactions  in  immune  sera.  These  points  will  be 
taken  up  in  the  description  of  the  individual  microorganisms,  and  will 
again  be  summarized  in  the  differential  tables  given  at  the  end  of  the 
chapters  dealing  with  this  group. 

BACILLUS  COLI  COMMUNIS  AND  MEMBERS  OF  THE  COLON 
BACILLUS    GROUP 

Under  the  name  of  " colon  bacilli"  are  grouped  a  number  of  varie- 
ties differing  from  one  another  in  minor  characteristics,  but  cor- 
responding in  certain  cardinal  points  which  stamp  them  as  close 
relatives  and  amply  warrant  their  consideration  under  one  heading. 
While  usually  growing  as  harmless  parasites  upon  the  animal  and  human 
body,  and  capable  of  leading  a  purely  saprophytic  existence,  they  may, 
nevertheless,  under  certain  circumstances  become  pathogenic  and  thus, 
both  culturally  and  in  their  pathological  significance,  form  a  link  between 
pure  saprophytes  like  Bacillus  lactis  aerogenes,  on  the  one  hand,  and 
the  more  strictly  pathogenic  Gram-negative  bacilli  of  the  paratyphoid, 
typhoid,  and  dysentery  groups,  on  the  other.  As  a  type  of  the  group 
we  may  consider  in  detail  its  most  prominent  and  thoroughly  studied 
member,  Bacillus  coli  communis. 

BACILLUS    COLI    COMMUNIS 

This  microorganism  was  seen  and  described  by  Buchner  1  in  1885. 
It  was  thoroughly  studied  in  the  years  immediately  following,  by  Esche- 
rich,2  in  connection  with  the  intestinal  contents  of  infants. 

Morphology. — Bacillus  coil  communis  is  a  short,  plump  rod  about 
1-3  micra  long,  and  varying  in  thickness  from  one-third  to  one-fifth 
of  its  length.  Under  varying  conditions  of  cultivation,  it  may  appear 
to  be  more  slender  than  this  or  shorter  and  even  coccoid  in  form.  In 
stained  preparations,  it  usually  appears  singly,  but  occasionally  may  be 
seen  in  short  chains.  It  stains  readily  with  the  usual  anilin  dyes  and 
decolorizes  by  Gram's  method.  Spores  are  not  formed.  It  is  motile, 
and  flagella  staining  reveals  eight  or  more  flagella  peripherally  arranged. 
Its  motility  is  subject  to  wide  variations.  Young  cultures,  in  the  first 

1  Buchner,  Arch.  f.  Hyg.,  3,  1885. 

*E8cherich,  "Die  Darmbakt.  des  Sauglinngs, "  Stuttgart,  1886;  Cent.  f.  Bakt., 
1,  1887. 


BACILLI   OF   THE   COLON-TYPHOID-DYSENTERY   GROUP     629 

generation  after  isolation  from  the  body,  may  be  extremely  motile, 
while  old  laboratory  strains  may  show  almost  no  motility.  Independent 
of  these  modifying  conditions,  however,  separate  races  may  show  indi- 
vidual characteristics  as  to  motilit\r,  varying  in  range  between  a  motility 
hardly  distinguishable  from  Brownian  movement  and  one  which  is  so 
active  as  to  be  but  little  less  than  that  of  the  typhoid  bacillus.  Ordi- 
narily, the  colon  bacillus  possesses  a  motility  intermediate  between 
these  two  extremes. 


FIG.  65. — BACCILLUS  COLI  COMMUNIS. 

Cultivation. — The  bacillus  is  an  aerobe  capable  of  anaerobic 
growth  under  suitable  cultural  conditions.  It  grows  well  on  the  sim- 
plest media  at  temperatures  ranging  from  20°  to  40°  C.,  but  finds  its 
optimum  growth  at  about  37.5°  C.  Upon  broth  it  grows  rapidly,  giving 
rise  to  general  clouding;  later  to  a  pellicle  and  a  light,  slightly  slimy 
sediment.  Within  moderate  ranges,  it  is  not  delicately  susceptible  to 
reaction,  growing  equally  well  on  media  slightly  acid  and  on  those  of  a 
moderate  alkalinity. 

Upon  agar,  it  forms  grayish  colonies  which  become  visible  within 
twelve  to  eighteen  hours,  gradually  becoming  more  and  more  opaque 
as  they  grow  older.  The  deep  colonies  are  dense,  evenly  granular,  oval, 
or  round.  Surface  colonies  often  show  a  characteristic  grape-leaf 


630 


PATHOGENIC   MICROORGANISMS 


structure,  or  may  be  round  and  flat,  and  show  a  definitely  raised,  glisten- 
ing surface.  Upon  agar  slants,  growth  occurs  in  a  uniform  layer. 

On  gelatin  the  colon  bacillus  grows  rapidly,  causing  no  liquefaction. 
Surface  colonies  are  apt  to  show  the  typical  grape-leaf  formation.  Deep 
colonies  are  round,  oblong,  and  glistening.  In  gelatin  stabs  growth  takes 
place  along  the  entire  line  of  inoculation,  spreading  in  a  thin  layer  over 
the  surface  of  the  medium. 

On  potato,  growth  is  abundant  and  easily  visible,  within  eighteen 


1  2  3 

FIG.  66. — BACCILLUS  COLT  COMMUNIS,  Grown  in:  1.  Dextrose,  2.  Lactose, 
3.  Saccharose  broth.  The  baccillus  forms  acid  and  gas  from  dextrose  and  lactose, 
not  from  saccharose.  Note  the  absence  of  growth  in  the  closed  arm  of  the  sac- 
charose tube,  in  which  no  acid  or  gas  is  formed. 

to  twenty-four  hours,  as  a  grayish-white,  glistening  layer  which  later 
turns  to  a  yellowish-brown,  and  in  old  cultures  often  to  a  dirty  green- 
ish-brown color. 

In  pepton  solution  indol  is  formed.  In  milk  there  is  acidity  and 
coagulation.  In  lactose-litmus-agar  acid  is  formed,  the  medium  becom- 
ing red,  and  gas-bubbles  appear  along  the  line  of  the  stab  inoculation. 

In  carbohydrate  broth,  pis  is  formed  in  dextrose,  lactose,  and  mannit, 
but  not  in  saccharose.  Levulose,  pilactose,  and  maltose  arc  also  fer- 
mented with  the  formation  of  acid  and  gas.  ' 


BACILLI    OF   THE    COLON-TYPHOID-DYSENTERY    GROUP     631 

Cultures  of  the  colon  bacillus  arc  characterized  by  a  peculiar  fetid 
odor  which  is  not  unlike  that  of  diluted  feces.  The  acids  formed  by  the 
colon  bacillus  from  sugars  are  chiefly  lactic,  acetic,  and  formic.  The 
gas  it  produces  consists  chiefly  of  CC>2  and  hydrogen.  The  bacillus 
grows  well  on  media  containing  urine  and  on  those  containing  bile. 
Upon  the  latter  fact  methods  for  the  isolation  of  the  colon  bacillus  from 
water  and  feces  have  been  based. 

Isolation  of  the  colon  bacillus  from  mixed  cultures  is  most  easily 
accomplished  by  plating  upon  lactose-litmus-agar,  the  Conradi-Drigal- 
ski  medium,  or  the  Endo  medium  after  preliminary  enrichment  if 
necessary  in  bile  or  malachite-green  broth.  (In  the  case  of  feces  such 
enrichment  is  superfluous.) 

Distribution. — The  colon  bacillus  is  a  constant  inhabitant  of  the 
intestinal  canal  of  human  beings  and  animals.  It  is  also  found  occasion- 
ally in  soil,  in  air,  in  water,  and  in  milk  and  is  practically  ubiquitous  in 
all  neighborhoods  which  are  thickly  inhabited.  When  found  in  nature 
its  presence  is  generally  taken  to  be  an  indication  of  contamination  from 
human  or  animal  sources.  Thus,  when  found  in  water  or  milk,  much 
hygienic  importance  is  attached  to  it.  Recently,  Papasotiriu3  and 
independently  of  him,  Prescott,4  have  reported  finding  bacilli  apparently 
identical  with  Bacillus  coli  upon  rye,  barley,  and  other  grains.  They 
believe,  upon  the  basis  of  this  discovery,  that  Bacillus  coli  is  widely 
distributed  in  nature  and  that  its  presence,  unless  it  appears  in  large 
numbers,  does  not  necessarily  indicate  recent  fecal  contamination. 
These  reports,  however,  have  not  found  confirmation  by  the  work  of 
others. 

In  man,  Bacillus  coli  appears  in  the  intestine  normally  soon  after 
birth,  at  about  the  time  of  taking  the  first  nourishment.5  From  this 
time  on,  throughout  life,  the  bacillus  is  a  constant  intestinal  inhabitant 
apparently  without  dependence  upon  the  diet.  Its  distribution  within 
the  intestine,  according  to  Gushing  and  Livingood,6  is  not  uniform,  it 
being  found  in  the  greatest  numbers  at  or  about  the  ileocecal  valve, 
diminishing  from  this  point  upward  to  the  duodenum  and  downward  as 
far  as  the  rectum.  Adami7  and  others  claim  that,  under  normal  con- 
ditions, the  bacillus  may  invade  the  portal  circulation,  possibly  by  the 

3  Papasotiriu,  Arch.  f.  Hyg.,  xli. 

4  Prescott,  Cent.  f.  Bakt.,  Eef.,  xxxiii,  1903. 

*  Schild,  Zeit.  f.  Hyg.,  xix,  1895;  Lemblce,  Arch.  f.  Hyg.,  xxiv,  1896. 
*Cushing    and    Livingood,    "Contributions    to    Med.    Sci.    by    Pupils    of    Wm. 
Welch,"  Johns  Hopk.  Press,  1900. 

7  Adami,  Jour,  of  Amer.  Med.  Assn.,  Dec.,  1899. 


632  PATHOGENIC  MICROORGANISMS 

intermediation  of  leucocytic  emigration  during  digestion.  After 
death,  at  autopsy,  Bacillus  coli  is  often  found  in  the  tissues  and  the  blood 
without  there  being  visible  lesions  of  the  intestinal  mucous  membrane.8 
It  is  probable,  also,  that  it  may  enter  and  live  in  the  circulation  a  few 
hours  before  death  during  the  agonal  stages. 

The  distribution  of  the  colon  bacilli  in  the  human  intestine  at  dif- 
ferent periods  of  life,  and  under  varying  dietetic  conditions  has  been 
considered  in  the  section  on  the  " normal  flora  of  the  intestinal  canal," 
p.  223. 

Extensive  investigations  have  been  carried  out  to  determine  whether 
or  not  the  constant  presence  of  this  microorganism  in  the  intestinal 
tract  is  an  indication  of  its  possessing  a  definite  physiological  function  of 
advantage  to  its  host.  It  has  been  argued  that  it  may  aid  in  the  fer- 
mentation of  carbohydrates.  The  question  has  been  approached  experi- 
mentally by  a  number  of  investigators.  Nuttall  and  Thierfelder  9 
delivered  guinea-pigs  from  the  mother  by  Cesarean  section  and  suc- 
ceeded in  preserving  them  from  infection  of  the  intestinal  canal  for 
thirteen  days.  Although  no  microorganisms  of  any  kind  were  found  in 
the  feces  of  these  animals,  no  harm  seemed  to  accrue  to  them,  and  some 
of  them  even  gained  in  weight.  Schottelius,10  on  the  other  hand, 
obtained  contradictory  results  with  chicks.  Allowing  eggs  to  hatch 
in  an  especially  constructed  glass  compartment,  he  succeeded  in 
keeping  the  chicks  and  their  entire  environment  sterile  for  seventeen 
days.  During  this  time  they  lost  weight,  did  not  thrive,  and  some 
of  them  were  moribund  at  the  end  of  the  second  week,  in  marked 
contrast  to  the  healthy,  well-nourished  controls,  fed  in  the  same  way, 
but  under  ordinary  environmental  conditions.  Although  insuffi- 
cient work  has  been  done  upon  this  important  question,  and  no  definite 
statement  can  be  made,  it  is  more  than  likely  that  the  function  of  the 
Bacillus  coli  in  the  intestine  is  not  inconsiderable  if  only  because  of  its 
possible  antagonism  to  certain  putrefactive  bacteria,  a  fact  which  has 
been  demonstrated  in  interesting  studies  by  Bienstock  n  and  others.12 

Pathogenicity. — The  pathogenicity  of  the  colon  bacillus  for 
animals  is  slight  and  varies  greatly  with  different  strains.  Intraperi- 
toneal  injections  of  1  c.c.  or  more  of  a  broth  culture  will  often  cause 
death  in  guinea-pigs.  Intravenously  administered  to  rabbits  it  may 

8  Birch-Hirschfeld,  Ziegler  's  Beitr.,  24,  1898. 

9  Nuttall  und  Thierf 'elder,  Zeit.  f .  Physiol.  Chemie,  xxi  and  xxii. 

10  Scltottelvus,  Arch.  f.  Hyg.,  xxiv,  1889. 

11  Bienstock,  Arch.  f.  Hyg.,  xxix,  1901. 

12  Tisser  and  Martelly,  Ann.  de  Pinst.  Pasteur,  1902. 


BACILLI   OF   THE    COLON-TYPHOID-DYSENTERY    GROUP     633 

frequently  cause  a  rapid  sinking  of  the  temperature  and  death  with 
symptoms  of  violent  intoxication  within  twenty-four  to  forty-eight 
hours.  Subcutaneous  inoculation  of  moderate  doses  usually  results  in 
nothing  more  than  a  localized  abscess  from  which  the  animals  recover. 

In  man,  a  large  variety  of  lesions  produced  by  Bacillus  coli  have 
been  described.  It  is  a  surprising  fact  that  disease  should  be  caused 
at  all,  in  man,  by  a  microorganism  which  is  so  constantly  present  in 
large  numbers  in  the  intestine  and  against  which,  therefore,  it  is  to  be 
expected  that  a  certain  amount  of  immunity  should  be  developed.  A 
number  of  explanations  for  this  state  of  affairs  have  been  advanced, 
none  of  them  entirely  satisfactory.  It  is  probable  that  none  of  the  poi- 
sonous products  of  the  colon  bacillus  are  absorbed  unchanged  by  the 
healthy  unbroken  mucosa  and  that,  therefore,  the  microorganisms  are> 
strictly  speaking,  at  all  times,  outside  of  the  body  proper.  Under  these 
circumstances,  no  considerable  process  of  immunization  would  be  antici- 
pated. It  is  also  possible  that,  whenever  an  infection  with  Bacillus  coli 
does  occur,the  infecting  organism  is  one  which  has  been  recently  acquired 
from  another  host,  having  no  specific  adaptation  to  the  infected  body. 
Virulence  may  possibly  be  enhanced  by  inflammatoryfprocesses  caused  by 
other  organisms.  Considering  the  subject  from  another  point  of  view, 
colon-bacillus  infection  may  possibly  take  place  simply  because  of 
unusual  temporary  reduction  of  the  resistance  of  the  host.  Whether  or 
not  altered  cultural  conditions  in  the  intestine  may  lead  to  marked 
enhancement  in  the  virulence  of  the  colon  bacilli  cannot  at  present  be 
decided.  The  opinion  has  been  frequently  advanced,  however,  without 
adequate  experimental  support. 

Septicemia,  due  to  the  colon  bacillus,  has  been  described  by  a  large 
number  of  observers.  It  is  doubtful,  however,  whether  many  of  these 
cases  represent  an  actual  primary  invasion  of  the  circulation  by  the 
bacilli,  or  whether  their  entrance  was  not  simply  a  secondary  phe- 
nomenon occurring  during  the  agonal  stages  of  another  condition.  A 
few  unquestionable  cases,  however,  have  been  reported,  and  there  can 
be  no  doubt  about  the  occurrence  of  the  condition,  although  it  is  prob- 
ably less  frequent  than  formerly  supposed.  The  writer  has  observed 
it  on  two  occasions  in  cases  during  the  lethal  stages  of  severe  systemic 
disease  due  to  other  causes.  An  extremely  interesting  group  of  such 
cases  are  those  occurring  in  new-born  infants,  in  which  generalized 
colon-bacillus  infection  may  lead  to  a  fatal  condition  known  as  WinckeFs 
disease  or  hemorrhagic  septicemia.13  Prominent  among  disease  processes 

13  Kamen,  Ziegler  '9  Beitr.,  U,  1896, 


634  PATHOGENIC   MICROORGANISMS 

attributed  to  these  microorganisms  are  various  diarrheal  conditions,  such 
as  cholera  nostras  and  cholera  infantum.  The  relation  of  these  maladies 
to  the  colon  bacillus  has  been  particularly  studied  by  Escherich,14 
but  satisfactory  evidence  that  these  bacilli  may  specifically  cause  such 
conditions  has  not  been  brought.  While  it  is  not  unlikely  that  under 
conditions  of  an  excessive  carbohydrate  diet,  colon  bacilli,  may  aggravate 
morbid  processes  by  a  voluminous  formation  of  gas,  they  do  not,  of 
themselves,  take  part  in  actual  putrefactive  processes.  It  is  likely, 
therefore,  that  in  most  of  the  intestinal  diseases  formerly  attributed 
purely  to  bacilli  of  the  colon  group,  these  microorganisms  actually  play 
but  a  secondary  part.15 

It  is  equally  difficult  to  decide  whether  or  not  these  bacilli  may  be 
regarded  as  the  primary  cause  of  peritonitis  following  perforation  of 
the  gut.  Although  regularly  found  in  such  conditions,  they  are  hardly 
ever  found  in  pure  culture,  being  accompanied  usually  by  staphylococci, 
streptococci,  or  other  microorganisms,  whose  relationship  to  disease  is 
far  more  definitely  established.  Isolated  cases  have  been  reported, 
however,  one  of  them  by  Welch,  in  which  Bacillus  coli  was  present  in 
the  peritoneum  in  pure  culture  without  there  having  been  any  intestinal 
perforation.16  Granting  that  the  bacillus  is  able  to  proliferate  within 
the  peritoneum,  there  is  no  reason  for  doubting  its  ability  of  giving  rise 
to  a  mild  suppurative  process. 

Inflammatory  conditions  in  the  liver  and  gall-bladder  have  fre- 
quently been  attributed  to  the  colon  bacillus.  It  has  been  isolated  from 
liver  abscesses,  from  the  bile,  and  from  the  centers  of  gall-stones.  Welch 
has  reported  a  case  of  acute  hemorrhagic  pancreatitis  in  which  the 
bacillus  was  isolated  from  the  gall-bladder  and  from  the  pancreas. 

In  the  bladder,  Bacillus  coli  frequently  gives  rise  to  cystitis  and 
occasionally  to  ascending  pyonephrosis.  No  other  microorganism,  in 
fact,  is  found  so  frequently  in  the  urine  as  this  one.  It  may  be  present, 
often,  in  individuals  in  whom  all  morbid  processes  are  absent.  The 
condition  is  frequently  observed  during  the  convalescence  from  typhoid 
fever.  It  may  disappear  spontaneously,  or  cystitis,  usually  of  a  mild, 
chronic  variety,  may  supervene. 

Localized  suppurations  due  to  this  bacillus  may  take  place  in  all 
parts  of  the  body.  They  are  most  frequently  localized  about  the  anus 
and  the  genitals,  and  are  usually  mild  and  amenable  to  the  simplest 
surgical  treatment. 

"Escherich,  loe.  cit. 

15  Herter,  "Bact.  Infec.  of  Digest.  Tract,"  N.  Y.,  1907. 

16  Welch,  Med.  News,  59,  1891. 


BACILLI   OF   THE   COLON-TYPHOID-DYSENTERY    GROUP      635 

For  a  consideration  of  the  distribution  of  colon  bacilli  in  the  intestines 
of  human  beings  at  various  ages  and  under  modified  dietary  conditions 
the  reader  is  referred  to  the  section  on  the  normal  flora  of  the  intestinal 
canal. 

Poisonous  Products  of  the  Colon  Bacillus. — The  colon  bacillus 
belongs  essentially  to  that  group  of  bacteria  whose  toxic  action  is  sup- 
posed to  be  due  to  the  poisonous  substances  contained  within  the 
bacillary  body.  Culture  filtrates  of  the  colon  bacillus  show  very  little 
toxicity  when  injected  into  animals;  whereas  the  injection  of  dead 
bacilli  produces  symptoms  almost  equal  in  severity  to  these  induced  by 
injection  of  the  live  microorganisms.  Corroborative  of  the  assumption 
of  this  endotoxic  nature  of  the  colon-bacillus  poison  is  the  fact  that, 
so  far,  no  antitoxic  bodies  have  been  demonstrated  in  serum  as  resulting 
from  immunization. 

Dead  colon  bacilli  have  a  very  high  toxicity  for  rabbits  and  some 
what  less  for  guinea  pigs. 

Immunization  with  the  Colon  Bacillus. — The  injection  into  animals 
of  gradually  increasing  doses  of  living  or  dead  colon  bacilli  gives  rise  to 
specific  bacteriolytic,  agglutinating,  and  precipitating  substances. 

The  bacteriolytic  substances  may  be  easily  demonstrated  by  the 
technique  of  the  Pfeiffer  reaction.  In  vitro  bacteriolysis  is  less  marked 
than  in  the  case  of  some  other  microorganisms  such  as  the  cholera  spiril- 
lum or  the  typhoid  bacillus.  Owing  probably  to  the  habitual  presence 
of  colon  bacilli  in  the  intestinal  tracts  of  animals  and  man,  considerable 
bacteriolysis  may  occasionally  be  demonstrated  in  the  serum  of  normal 
individuals. 

Agglutinins  for  the  colon  bacillus  have  often  been  produced  in  the 
sera  of  immunized  animals  in  concentration  sufficient  to  be  active  in 
dilutions  of  1  :  5000  and  over.  The  agglutinins  are  produced  equally 
well  by  the  injection  of  live  cultures  and  of  those  killed  by  heat,  if  the 
temperature  used  for  sterilization  does  not  exceed  100°  C.  It  is  17 
a  noticeable  fact  that  the  injection  of  any  specific  race  of  colon  bacilli 
produces,  in  the  immunized  animal,  high  agglutination  values  only  for 
the  individual  culture  used  for  immunization,  while  other  strains  of 
colon  bacilli,  although  agglutinated  by  the  serum  in  higher  dilution 
than  are  paratyphoid  or  typhoid  bacilli,  require  much  higher  concen- 
tration than  does  the  original  strain.  The  subject  has  been  extensively 
studied  by  a  number  of  observers  and  illustrates  the  extreme  individual 
specificity  of  the  agglutination  reaction.  Thus  a  serum  which  will 

"  Wolff,  Cent.  f.  Bakt.,  xxv,  1899. 


636  PATHOGENIC  MICROORGANISMS 

agglutinate  its  homologous  strains  in  dilutions  of  one  to  1000  will  often 
fail  to  agglutinate  other  races  of  Bacillus  coli  in  dilutions  of  1  :  500 
or  1  :  600. 

The  normal  serum  of  adult  animals  and  man  will  often  agglutinate 
this  bacillus  in  dilutions  as  high  as  1  :  10  or  1  :  20 — a  phenomenon  pos- 
sibly referable  to  its  habitual  presence  within  the  body.  Corroborating 
this  assumption  is  the  observation  of  Kraus  and  Low,18  that  the  serurn 
of  new-born  animals  possesses  no  such  agglutinating  powers.  The 
fact  that  agglutinins  for  the  colon  bacillus  are  increased  in  the  serum 
of  patients  convalescing  from  typhoid  fever  or  dysentery  is  probably 
to  be  explained,  partly  by  the  increase  of  the  group  agglutinins  pro- 
duced by  the  specific  infecting  agent,  and  partly  by  the  invasion  of 
colon  bacilli,  or  the  absorption  of  its  products  induced  by  the  diseased 
state  of  the  intestinal  mucous  membrane. 

Varieties  of  the  Colon  Bacillus. — During  the  earlier  days  of  bac- 
teriological investigations,  a  large  number  of  distinct  varieties  of  colon 
bacilli  were  described,  many  of  which  may  now  be  dismissed  as  based 
simply  upon  a  temporary  depression  of  one  or  another  cultural  charac- 
teristic of  Bacillus  coli  communis,  while  others  can  be  definitely  included 
within  other  closely  related,  but  distinct  groups. 

That  secondary  features,  such  as  dimensions,  motility,  and  luxuri- 
ance of  growth  upon  various  media,  may  be  markedly  altered  by  arti- 
ficial cultivation  is  a  common  observation.  It  has  not,  however,  been 
satisfactorily  shown  that  cardinal  characteristics,  such  as  the  forma- 
tion of  indol  from  pepton,  or  the  power  to  produce  gas  from  dextrose 
and  lactose,  can  be  permanently  suppressed  without  actual  injury  or 
inhibition  of  the  normal  vitality  of  the  microorganism.  Such  alter- 
ation is,  in  fact,  contrary  to  experience,  which  demonstrates  that 
whenever  such  changes  do  occur,  they  are  purely  temporary  and  a  few 
generations  of  cultivation  under  favorable  environmental  conditions 
will  regularly  restore  the  organism  to  its  normal  activity. 

Distinct  and  constant  varieties  of  the  Colon  Bacillus  or,  at  least, 
close  biological  relatives  do  occur.  It  is  necessary  to  consider  the  organ^ 
isms  as  a  group  for  this  reason,  since,  in  sanitary  work,  it  is  of  the  utmost 
importance  to  recognize  forms  which  should  properly  be  classified  under 
this  category.  It  may  be  well,  therefore,  to  reiterate  the  criteria  for 
identification  of  the  group  established  by  the  American  Public  Health 
Association  Committee  on  Standard  Methods  of  Water  Analysis.19 
This  report  defines  the  general  characteristics  of  the  group,  as  follows: 

15  Kraus  und  Low,  Wien.  klin.  Woch.,  1899. 


BACILLI   OF   THE   COLON-TYPHOID-DYSENTERY   GROUP     637 

short  bacillus  form,  failure  of  spore  formation,  facultative  anaerobiosis, 
growth  on  gelatin  without  liquefaction  in  fourteen  days,  Gram-negative 
stain  and  fermentation  of  dextrose  and  lactose  with  gas  formation. 
They  add  that  there  is  a  positive  reaction  with  esculin. 

The  organisms  which  are  placed  under  the  Colon  group  in  this  report 
are  the  B.  Coli  communis  described  above,  the  B.  Coli  communior 
of  Durham,  the  B.  aerogenes  of  Escherich,  and  B.  acidi-lactici  of  Htieppe. 
The  chief  differential  characteristics  are  as  follows : 


Dextrose. 

Lactose. 

Saccharose. 

Dulcit. 

B   coli  communis  

+ 

+ 

+ 

B  coli  communior 

4- 

, 

, 

, 

B.  lact.  aerogenes  
B.  lact.  acidi  

t 

t 

- 

Individual  descriptions  of  these  organisms  follow: 

B.  COLI  COMMUNIOR. — This  organism  first  described  by  Durham 
was  called  Communior  by  him  because  of  his  belief  that  it  was  more 
abundant  in  the  human  and  animal  intestine  than  the  Communis  type. 
It  possesses  all  the  characteristics  of  the  Colon  group.  It  is  a  Gram- 
negative  bacillus,  motile,  non-sporulating,  and  morphologically  indis- 
tinguishable from  the  Communis  variety.  It  does  not  liquefy  gelatin, 
it  produces  indol  from  pepton,  coagulates  and  acidifies  milk,  and  grows 
characteristically  upon  agar  and  potato.  It  differs  from  B.  coli  com- 
munis in  that  it  produces  acid  and  gas  from  saccharose  as  well  as  from 
dextrose  and  lactose,  whereas  the  former  does  not  form  acid  or  gas  from 
saccharose.  Several  varieties  have  been  described  by  Melia,  and  by 
A  very.  The  Melia  type  differs  from  the  ordinary  variety  in  not  pro- 
ducing indol.  The  Avery  type  did  not  coagulate  milk. 

B.  LACTIS  AEROGENES. — Bacillus  lactis  aerogenes  is  the  type  of  a 
group  which  is  closely  similar  to  the  colon  group  and  often  distin- 
guished from  it  with  difficulty.  It  was  first  described  in  1885  by 
Escherich  19  who  isolated  it  from  the  feces  of  infants.  Since  then  it  has 
been  learned  that  this  bacillus  is  almost  constantly  present  in  milk, 
and,  together  with  one  or  two  other  microorganisms,  is  the  chief  cause 
of  the  ordinary  souring  of  milk.  Apart  from  its  occurrence  in  milk, 
moreover,  the  bacillus  is  widely  distributed  in  nature,  being  found  in 
feces,  in  water,  and  in  sewage. 

19  A.  P.  H.  A.  Standard  Methods  of  Water  Analysis,  1915. 


638 


PATHOGENIC  MICROORGANISMS 


It  is  distinguished  from  the  Colon  bacillus  chiefly  by  the  fact  that  it 
is  less  motile,  hardly  ever  forms  chains,  and,  when  cultivated  upon 
suitable  media,  especially  milk,  it  possesses  a  distinct  capsule.  It 
differs  from  other  forms  of  the  Colon  group  in  not  fermenting 
dulcite,  and  differs  from  B.  acidi-lactici  in  fermenting  saccharose.  It 
ferments  with  gas  production,  dextrose,  lactose,  saccharose,  mannite 
and  raffinose.  It  produces  indol,  reduces  nitrate,  possesses  either 
no  motility,  or  is  very  slightly  motile.  It  coagulates  milk,  and  when 
grown  on  milk  or  lactose  bile  it  often  makes  a  stringy  viscous  cult- 
ure, On  agar  and  gelatin  it  makes  heavy  white  colonies  of  a  some- 


1  2  3 

FIG.    67. — BACILLUS    COLI  COMMUNIOR.     Grown  in:    1.   Dextrose,   2.   Lactose,  3, 

Saccharose  broth. 

what  mucoid  appearance,  certainly  more  mucoid  than  most  colon 
colonies.  It  does  not  liquefy  gelatin.  In  broth  it  causes  general  cloud- 
ing with  later  a  pellicle,  and  a  sour  odor.  It  grows  heavily  on  potato. 

It  is  a  facultative  anaerobe  and  grows  at  room  temperature. 

Varieties  have  been  described  depending  upon  minor  cultural  char- 
acteristics which  have  no  particular  importance  in  this  connection. 

The  pathogenicity  of  Bacillus  lactis  aerogenes  for  man  is  slight. 
Its  chief  claims  to  importance  lie  in  its  milk-coagulating  properties 
and  its  almost  constant  presence  in  the  human  intestine.  In  infants,  it 
may  give  rise  to  flatulence  and  it  has  been  occasionally  observed  as  the 
sole  incitant  of  cystitis.  Among  such  cases  rare  instances  have  been 


BACILLI   OF   THE    COLON-TYPHOID-DYSENTERY   GROUP     639 

observed  in  which  it  has  formed  gas  in  the  bladder  (pneumaturia). 
When  this  occurs  the  urine  is  not  ammoniacal  but  remains  acid. 

Different  strains  of  this  bacillus  vary  much  in  their  pathogenicity 
for  animals.  Wilde  claims  that  it  is  more  pathogenic  for  white  mice  and 
guinea-pigs  than  is  the  bacillus  of  Friedlander.  He  speaks  of  it  as  the 
most  virulent  member  of  this  group.  Kraus,  writing  in  Fluegge's 
"Mikroorganismen,"  rates  its  pathogenicity  less  high. 

Closely  related  to  this  bacillus,  as  well  as  those  of  the  Friedlander 
group,  is  an  encapsulated  bacillus  isolated  from  a  case  of  broncho- 
pneumonia  by  Mallory  and  Wright,20  which  is  strongly  pathogenic  for 
mice,  guinea-pigs,  and  rabbits. 

B.  ACIDI-LACTICI. — This  organism,  like  the  others,  is  a  Gram- 
negative,  non-liquefying,  non-sporulating  bacillus.  Just  like  the  B. 
aerogenes,  it  has  no  motility.  It  differs  from  the  Colon  bacilli  proper 
in  not  fermenting  dulcite.  It  differs  from  the  Lactis  aerogenes  in 
failing  to  ferment  saccharose.  Like  the  Lactis  aerogenes,  it  is  non- 
motile.  It  forms  indol,  reduces  nitrate,  and  coagulates  milk. 

It  is  commonly  present  in  milk  and  may  be  present  in  water.  It  is 
often  found  in  the  intestinal  canal  but  as  far  as  we  know  has  no  patho- 
genic significance. 

BACILLUS  FECALIS  ALKALIGENES. — In  1896  Petruschky21  described 
a  bacillus  which  is  a  not  infrequent  inhabitant  of  the  human  intestine, 
being  found  chiefly  in  the  lower  part  of  the  small  intestine  and  in  the  large 
intestine.  This  organism,  which  he  called  Bacillus  fecalis  alkaligenes, 
is  of  little  pathogenic  importance,  although  Neufeld  states  that  he  has 
seen  a  case  of  severe  gastroenteritis  in  which  the  watery  defecations 
contained  this  bacillus  in  almost  pure  culture.  As  a  rule,  however,  this 
organism  cannot  be  regarded  as  pathogenic,  and  is  important  chiefly 
because  of  the  ease  with  which  it  may  be  mistaken  for  Bacillus  typhosus. 

Bacillus  fecalis  alkaligenes  is  an  actively  motile,  Gram-negative 
bacillus,  possessing,  like  the  typhoid  bacillus,  numerous  peritrichal 
flagella.  On  the  ordinary  culture  media  it  grows  like  the  typhoid 
bacillus.  It  does  not  coagulate  milk.  It  produces  no  indol,  and  on 
sugar  media  in  fermentation  tubes  produces  no  acid  or  gas.  On  potato, 
its  growth,  while  somewhat  heavier  than  that  of  the  typhoid  bacillus, 
is  not  sufficiently  so  to  permit  easy  differentiation.  It  differs  from 
Bacillus  typhosus  in  that  it  produces  no  acid  on  any  of  the  sugar  media, 
and  is  therefore  easily  differentiated  by  cultivation  upon  Hiss  serum- 

20  Mallory  and  Wright,  Zeit.  f.  Hyg.,  20,  1895. 

21  Petruschky,  Cent.  f.  Bakt.,  I,  ixix,  1896. 


640  PATHOGENIC   MICROORGANISMS 

water  media  or  on  pep  ton  waters  containing  sugars.  On  the  Hiss 
semi-solid  tube-medium  Bacillus  fecalis  alkaligenes,  while  clouding 
the  medium  throughout,  grows  most  heavily  on  the  surface,  where, 
eventually,  it  forms  a  pellicle, 

BACILLI  OF  THE  PROTEUS  GROUP 

There  are  a  great  many  other  organisms  which  are  similar  to  the 
Colon  Bacillus  in  general  appearance  and  superficial  morphological  and 
cultural  characteristics,  and  which  are  found  frequently  associated  with 
it  in  feces,  water  and  sewage.  It  will  be  important  for  this  reason  to 
speak  of  them  briefly.  The  most  important  of  these  is  the  Proteus 
Group,  which  is  sharply  separable  from  the  Colon  and  allied  bacteria 
by  its  gelatin  liquefaction. 

The  bacilli  of  this  group  have  little  pathological  interest,  but  are 
important  because  of  the  frequency  with  which  they  are  encountered  in 
routine  bacteriological  work.  They  may  confuse  the  inexperienced 
because  of  a  superficial  similarity  to  bacilli  of  the  colon-typhoid  group. 
In  form  they  may  be  short  and  plump  or  long  and  slender,  staining  easily 
with  anilin  dyes  and  decolorizing  with  Gram's  method.  They  are 
actively  motile  and  possess  many  flagella.  Individuals  stain  irregularly, 
often  showing  unstained  areas  near  the  center.  The  so-called  Bacillus 
proteus  vulgaris  described  by  Hauser  22  in  1885  is  the  type  of  the  group. 

Bacilli  of  this  group  are  widely  distributed,  being  found  in  water, 
soil,  air,  and  wherever  putrefaction  takes  place.  In  fact,  proteus  is  one 
of  the  true  putrefactive  bacteria  possessing  the  power  to  cause  the  cleav- 
age of  proteins  into  their  simplest  radicles. 

Bacillus  proteus  vulgaris  grows  best  at  temperatures  at  or  about 
25°  C.  and  develops  upon  the  simplest  media.  It  is  a  facultative 
anaerobe  and  forms  no  spores.  In  broth,  it  produces  rapid  clouding  with 
a  pellicle  and  the  formation  of  a  mucoid  sediment. 

In  gelatin,  the  colonies  are  characteristically  irregular,  giving  the 
name  to  this  group. 

Gelatin  is  Rapidly  Liquefied. — Liquefaction,  however,  is  diminished 
or  even  inhibited  under  anaerobic  conditions. 

On  agar  and  other  solid  media,  as  well  as  upon  gelatin  before  lique- 
faction has  taken  place,  characteristic  colonies  are  produced.  From 
the  central  flat,  grayish-white  colony  nucleus,  numerous  irregular 
streamers  grow  out  over  the  surrounding  media,  giving  the  colony  a 
stellate  appearance.  On  potato,  it  forms  a  dirty,  yellowish  growth. 

22  Hauser,  "Ueber  Faulniss-Bakt., ' '  Leipzig,  1885. 


BACILLI    OF    THE    COLON-TYPHOID-DYSENTERY    GROUP     041 

In  milk,  there  is  coagulation  and  an  acid  reaction  at  first;  later  the 
casein  is  redissolved  by  proteolysis.  Blood  serum  is  often  liquefied, 
but  not  by  all  races. 

A  great  many  really  dissimilar  bacteria  have  been  described  under 
the  name  of  Proteus.  The  type  of  the  group  is  the  so-called  Proteus 
vulgaris  (Hauser,  1885).  Other  organisms  spoken  of  as  proteus  are  the 
Proteus  mirabilis,  which  differs  in  slower  gelatin  liquefaction  from 
vulgaris,  the  Proteus  Zenkeri,  which  does  not  liquefy  gelatin,  the  Proteus 
septicus,  and  the  Bacillus  Zopfi,  a  Gram-positive  organism.  A  good 
many  of  these  were  formerly  classified  as  of  Bacterium  termo.  Closely 
related  is  the  slow  liquefying  organism  known  as  Bacillus  cloacce,  com- 
mon in  sewage. 

There  is  no  group  which  so  urgently  requires  study  as  this,  since 
organisms  belonging  here  are  so  often  found  in  the  human  body  and 
human  excreta.  In  urine  we  have  encountered  a  non-gelatin  liquefying 
Gram-negative  bacillus  belonging  to  this  group  which  has  given  us 
much  trouble  in  identification.  As  far  as  we  can  establish  any  general 
characteristics  for  the  group  at  all,  we  may  say  that  they  are  Gram- 
negative,  non-spore-bearing,  motile  bacilli,  which  on  the  surface  of 
gelatin  plates  show  colonies  characterized  by  spreading  streamers, 
most  of  which  liquefy  gelatin,  a  few  of  which,  however,  do  not.  All 
of  them  ferment  dextrose  and  saccharose  with  gas,  but  few  of  them 
attack  lactose. 

The  pathogenic  powers  of  proteus  are  slight.  Large  doses  injected 
into  animals  may  give  rise  to  localized  abscesses.  In  man  proteus 
infections  have  been  described  in  the  bladder,  in  most  cases,  however, 
together  with  some  other  microorganism.  The  Urobacillus  lique- 
faciens  septicus  described  by  Krogius  was  a  variety  of  this  group. 
Epidemics23  of  meat  poisoning  have  been  attributed  to  the  proteus 
family  by  some  observers.  Thus  Wesenberg24  cultivated  a  proteus 
from25  putrid  meat  which  had  caused  acute  gastroenteritis  in  sixty- 
three  individuals.  Similar  epidemics  have  been  reported  by  Silber- 
schmidt,25  Pfuhl,26  and  others. 

B.  CLOACAE. — This  organism  was  first  described  by  Jordan  and  is 
one  of  the  commonest  of  the  sewage  bacteria.  It  is  closely  related  to 
the  Proteus  organisms,  but  is  less  motile  than  they.  It  coagulates  milk, 
and  liquefies  gelatin,  but  its  gelatin  liquefaction  is  not  as  active  as  that 

X8chniteler,  Cent.  f.  Bakt.,  viii,  1890. 

24  Wesenberg,  Zeit.  f .  Hyg.,  xxviii,  1898. 

25  Silberschmidt,  Zeit.  f.  Hyg.,  xxx,  1899. 
29  Pfuhl,  Zeit.  f.  Hyg.,  xxxv,  1900. 


642  PATHOGENIC  MICROORGANISMS 

of  the  Proteus  group.  It  forms  indol  and  produces  acid  and  gas  on 
dextrose  and  saccharose,  but  one  of  its  chief  characteristics  is  its  slight 
action  on  lactose.  Jordan  states  as  one  of  its  chief  characteristics  the 
relatively  large  proportion  of  C02  formed  as  compared  with  hydrogen, 
the  ratio  being  in  some  cases  as  high  as  5  to  1.  Kendall,  Day  and 
Walker27  have  observed  the  same  thing.  The  same  investigators 
state  that  after  three  days'  growth,  even  sugar  broths  become  alkalin, 
owing  to  protein  decomposition. 

Recently  certain  stains  of  Proteus  have  become  important  because 
of  their  apparently  specific  agglutination  in  Typhus  Serum  (Weil- 
Felix  Reaction).  See  chapter  on  Typhus. 

Were  we  following  a  purely  biological  order  of  presentation,  we 
should  now  proceed  to  a  description  of  the  organisms  belonging  to  the 
so-called  Mucosus  Capsulatus  or  Friedlander  group.  These  bacilli 
are  closely  related  to  the  Colon  type,  more  particularly  to  the  B.  aero- 
genes  variety,  and  have  been  regarded  by  some  observers,  notably 
Fitzgerald,  as  perhaps  representing  members  of  the  Colon  group  which 
have  acquired  capsulation  and  virulence.  In  practice,  however,  these 
bacteria  are  rarely  encountered  under  conditions  where  differentiation 
from  Colon  bacilli  is  necessary,  and  their  heavy  mucoid  colonies  and 
capsulated  morphology  renders  their  recognition  relatively  easy.  It 
will  be  better,  therefore,  from  the  point  of  view  of  practical  discussion, 
to  proceed  directly  to  the  study  of  organisms  of  the  typhoid  and  dysen- 
tery groups,  since  these  are  the  ones  which  in  medical  and  sanitary 
bacteriology,  are  associated  in  the  human  body,  in  water  and  sewage 
with  members  of  the  Colon  group  and  which,  therefore,  present  the  most 
frequent  differential  problems. 

27  Kendall,  Day  and  Walker,  Jour.  Amer.  Chem.  Soc.,  1913,  35. 


CHAPTER  XXXII 

BACILLI  OF  THE  COLON-TYPHOID-DYSENTtfRY  GROUP      (Continued) 
THE  BACILLUS  OF  TYPHOID  FEVER 

(Bacillus  typhosus,  Bacillus  typhi  abdominalis) 

TYPHOID  FEVER,  because  of  its  wide  distribution  and  almost  con- 
stant presence  in  most  communities,  has  from  the  earliest  days  been  the 
subject  of  much  etiological  inquiry.  A  definite  conception  as  to  its 
infectiousness  and  transmission  from  case  to  case  was  formed  as  early 
as  1856  by  Budd.1 

But  it  was  not  until  1880  that  Eberth  2  discovered  in  the  spleen  and 
mesenteric  glands  of  typhoid-fever  patients  who  had  come  to  autopsy, 
a  bacillus  which  we  now  know  to  be  the  cause  of  the  disease.  Final 
proof  of  such  an  etiological  connection  was  then  brought  by  Gaffky,3 
who  not  only  saw  the  bacteria  referred  to  by  Eberth,  but  succeeded  in 
obtaining  them  in  pure  culture  and  studying  their  growth  characteristics. 

Morphology  and  Staining1. — The  typhoid  bacillus  is  a  short  rod 
from  1-3. 5ju  in  length  with  a  varying  width  of  from  .5  to  .8/x.  In  appear- 
ance it  has  nothing  absolutely  distinctive  which  could  serve  to  differen- 
tiate it  from  other  bacilli  of  the  typhoid-colon  group,  except  that  it  has 
a  general  tendency  to  greater  slenderness.  Its  ends  are  rounded  without 
ever  being  club-shaped.  Contrary  to  the  descriptions  of  the  earlier 
observers,  typhoid  bacilli  do  not  form  spores.  They  are  actively  motile 
and  have  twelve  or  more  flagella  peripherally  arranged. 

The  bacilli  stain  readily  with  the  usual  anilin  dyes.  Stained  by 
Gram's  method,  they  are  decolorized. 

Cultivation. — Bacillus  typhosus  is  easily  cultivated  upon  the 
usual  laboratory  media.  It  is  not  delicately  susceptible  to  reaction,  but 
will  grow  well  upon  media  moderately  alkaline  or  acid.  It  is  an  aerobic 
and  facultative  anaerobic  organism,  when  the  proper  nutriment  is 
present.  Upon  agar  plates  growth  appears  within  eighteen  to  twenty- 

lBudd,  "Intestinal  Fever,"  Lancet,  1856. 
2  Eberth,  Virch.  Archiv.,  81,  1880,  and  83,  1881. 
?  Gaffky,  Mitt.  a.  d.  kais.  Gesundheitsamt,  2,  1884. 

643 


644 


PATHOGENIC  MICROORGANISMS 


FIG.  68. — BACILLUS  TYPHOSDS,  from  twenty-four-hour  culture  on  agarl 


FIG.  69.— BACILLUS  TYPHOSUS,  showing  flagella.      (After  Frankel  and  Pfeiffer. 


BACILLI   OF   THE   COLON-TYPHOID-DYSENTERY   GROUP     645 

four  hours  as  small  grayish  colonies  at  first  transparent,  later  opaque. 
Upon  agar  slants  growth  takes  place  in  a  uniform  layer.  There  is 
nothing  characteristic  about  this  growth  to  aid  in  differentiation. 

In  broth,  the  typhoid  bacillus  grows  rapidly,  giving  rise  to  an  even 
clouding,  rarely  to  a  pellicle. 

Upon  gelatin,  the  typhoid  bacillus  grows  readily  and  does  not  liquefy 
the  medium.  In  stabs,  growth  takes  place  along  the  entire  extent 
of  the  stab  and  over  the  surface  of  the  gelatin  in  a  thin  layer.  In  gelatin 
plates  the  growth  may  show  some  differences  from  that  of  other  mem- 
bers of  this  group,  and  this  medium  was  formerly  much  used  for  isolation 
of  the  bacillus  from  mixed  cultures,  Growth  appears  within  twenty- 


FIG.  70. — SURFACE  COLONY  OF  BACILLUS  TYPHOSUS  ON  GELATIN.      (After  Heim.) 

four  hours  as  small  transparent,  oval,  round,  or  occasionally  leaf-shaped 
colonies  which  are  smaller,  more  delicate,  and  more  transparent  than 
contemporary  colonies  of  the  colon  bacillus.  They  do  not,  however, 
show  any  reliable  differential  features  from  bacilli  of  the  dysentery 
group.  As  the  colonies  grow  older  they  grow  heavier,  more  opaque,  and 
lose  much  of  their  early  differential  value. 

On  potato  the  growth  of  typhoid  bacilli  is  distinctive,  and  this  medium 
was  recommended  by  Gaffky4  in  his  early  researches  for  purposes  of 
identification.  On  it  typhoid  bacilli,  after  twenty-four  to  forty-eight 
hours,  produce  a  hardly  visible  growth,  evident  to  the  naked  eye  only 

4  Gaffky,  loc.  cit. 


646  PATHOGENIC   MICROORGANISMS 

by  a  slight  moist  glistening,  an  appearance  which  is  in  marked  contrast 
to  the  grayish-yellow  or  even  brown  and  abundant  growth  of  colon 
bacilli.  If  the  potato  medium  is  rendered  neutral  or  alkaline,  this 
distinction  disappears,  the  typhoid  bacillus  growing  more  abundantly. 

In  milk,  typhoid  bacilli  do  not  produce  coagulation.  In  litmus-milk, 
during  the  first  twenty-four  hours,  the  color  is  changed  to  a  reddish  or 
violet  tinge  by  the  formation  of  acid  from  the  small  quantities  of  mono- 
saccharid  present.  Later  the  color  becomes  deep  blue  owing  to  the 
formation  of  alkali. 

In  Dunham's  pepton  solution  no  indol  is  produced.  According  to 
Peckham,  however,  continuous  cultivation  in  rich  pepton  media  may 
lead  to  eventual  indol  formation  by  typhoid  bacilli.  This  fact  has  no 
bearing  on  the  value  of  the  indol  test,  as  indol  is  never  have  formed 
under  the  usual  cultural  conditions. 

Tested  for  its  power  to  form  acid  from  sugars  commonly  used  in 
differential  tests,  typhoid  bacilli  form  add,  but  no  gas,  on  the  mono- 
saccharides,  on  mannit,  maltose  and  dextrin,  and  neither  add  nor  gas  on 
lactose  and  saccharose.  (See  Table,  p.  718.) 

In  the  Hiss  tube  medium  (formerly  employed  extensively)  the 
typhoid  bacillus  within  eighteen  to  twenty-four  hours  produces  an  even 
clouding  by  virtue  of  its  motility,  but  does  not  form  gas.  In  contradis- 
tinction to  this,  dysentery  bacilli  grow  only  along  the  line  of  inocula- 
tion, while  bacilli  of  the  colon  group  move  in  irregular  sky-rocket-like 
figures  away  from  the  stab,  at  the  same  time  breaking  up  the  medium 
by  the  formation  of  gas-bubbles.  Some  actively  motile  colon  bacilli 
cloud  the  medium,  but  the  ruptures  caused  by  the  gas  are  always 
evident. 

The  differentiation  of  the  typhoid  bacillus  in  pure  culture  from  similar 
microorganisms  by  means  of  its  growth  upon  media  has  been  the  sub- 
ject of  many  investigations.  It  is  neither  practicable  nor  desirable  to 
enumerate  all  the  various  media  which  have  been  devised  and  reported. 
The  aim  has  been  chiefly  the  differentiation  of  typhoid  bacilli  from  the 
bacilli  of  the  colon  group,  and  most  of  the  media  have  been  devised  with 
this  end  in  view.  (See  section  on  Media.) 

Rothberger  5  devised  a  mixture  of  glucose  agar  to  which  is  added 
1  per  cent  of  a  saturated  aqueous  solution  of  neutral-red.  Shake- 
cultures  or  stab-cultures  are  made  in  tubes  of  this  medium.  The  typhoid 
bacillus  causes  no  changes  in  it,  while  members  of  the  colon  group,  by 

5  Rothberger,  Cent,  f .  Bakt.,  xxiv,  1898. 


BACILLI   OF   THE    COLON-TYPHOID-DYSENTERY    GROUP     647 

reduction  of  the  neutral-red,  decolorize  the  medium  and  produce  gas  by 
fermentation  of  the  sugar. 

Utilizing  the  fact  that  bile-salts  are  precipitated  in  the  presence  of 
acids,  MacConkey  devised  a  medium  composed  of  sodium  glycocholate, 
pepton,  lactose,  and  agar  (the  composition  of  this  medium  is  given  on 
page  164),  in  which  Bacillus  typhosus  grows  without  causing  such 
change,  but  distinct  clouding  results  from  the  growth  of  the  colon 
bacillus  which,  producing  acid  from  the  lactose,  causes  precipitation  of 
the  bile-salts. 

On  Wurtz's  lactose-litmus-agar  (see  page  153)  the  typhoid  bacillus 
produces  no  acid,  but  eventually  deepens  the  purple  color  to  blue; 
the  colon  bacillus  produces  acid  and  in  stab-cultures  gas  bubbles  and 
the  color  changes  to  red. 

In  Barsiekow's  (see  page  164)  lactose-nutrose-litmus  mixture  the 
typhoid  bacillus  causes  no  change,  while  the  colon  bacillus  produces 
coagulation  and  an  acid  reaction. 

The  differentiation  of  the  typhoid  bacillus  from  other  similar  organ- 
isms of  the  typhoid,  dysentery,  colon  group,  is  based  chiefly  on  growth, 
upon  differential  media  in  which  the  inability  of  the  typhoid  bacillus 
to  form  acid  or  gas  from  lactose  has  been  the  most  commonly  used  basis 
for  differentiation.  Various  indicators  to  show  whether  acid  has  been 
formed,  added  to  such  media  will  sharply  separate  this  organism  from 
the  colon  bacilli  and  their  close  relatives.  Failure  to  produce  gas  with 
dextrose,  differentiates  it  from  the  paratyphoid  group.  The  reader  is 
referred  to  the  differential  tables  given  on  page  687  and  718,  for  the 
basic  reactions  upon  which  cultural  differentiation  is  made.  The 
media  most  convenient  for  this  purpose  are,  in  plates,  the  Conradi- 
Drigalski  medium,  the  Endo  medium,  the  Krumwiede  brilliant  green 
medium,  or  the  Teague  eosin-methylene-blue  medium,  all  of  which  are 
described  in  the  section  on  media;  and,  in  tubes,  some  of  the  most 
convenient  media  are  the  Hiss  semi-solid  mentioned  above,  Barsiekow's 
medium,  or  the  Russell  double  sugar  agar.  The  Russell  double  sugar 
agar  is  particularly  useful  to  give  a  quick  index  of  differentiation,  since 
it  contains  both  lactose  and  glucose,  and,  whereas,  the  colon  group  give 
redness  throughout,  and  a  few  gas  bubbles,  the  typhoid  gives  no  gas,  a 
red  butt  due  to  its  action  in  the  depths  of  the  stab  on  the  glucose  and 
an  uncolored  surface  growth. 

Final    differentiation   is    best    based    upon    specific    agglutination. 


Winsloir,  Klu/lcr  and  Hothenberg,  Jour,  of  Bacter.,  4,  1919,  426. 


648  PATHOGENIC   MICROORGANISMS 

Winslow,  Kligler  and  Rothberg,6  on  the  basis  of  recent  careful  invests 
gations,  describe  the  typhoid  bacillus  as  a  Gram-negative,  non-spore 
forming,  actively  motile  rod  which  forms  translucent  irregular  colonies 
on  gelatin,  and  a  colorless  growth  on  potato.  It  produced  strong  and 
prompt  acid,  but  no  gas,  on  media  containing  the  hexoses,  maltose, 
mannit,  sorbit,  xylose  (rapid  or  slow),  and  dextrin;  it  does  not  attack 
arabinose,  rhamnose,  or  lactose;  produces  a  slight  initial  reddening  of 
litmus  milk,  which,  after  two  weeks,  reverts  to  neutrality  or  slight  alka- 
linity. It  does  not  form  indol,  nor  liquefy  gelatin,  does  not  grow  in 
asparagin-mannitol  medium,  does  not  reduce  neutral  red,  and  causes 
browning  of  lead  acetate  medium  (irregular) .  It  has  low  tolerance  for 
acid,  but  high  tolerance  for  malachite  and  brilliant  green  dyes.  It  has 
characteristic  serum  agglutination. 

Differences  Within  the  Typhoid  Group. — Recent  work  has  shown 
that  not  all  typhoid  bacilli  are  culturally  alike,  there  being  two  distinct 
groups,  one  which  ferments  xylose  rapidly,  the  other  slowly.  Since 
there  are  also  antigenic  differences  it  may  be  necessary  in  the  future 
to  speak  rather  of  a  typhoid  group  than  of  the  typhoid  bacillus. 

Xylose  fermentations  of  typhoid  bacilli  have  recently  been  studied 
in  more  detail  by  Krumwiede,  Kohn  and  Valentine,7  and  by  Morishima.8 
The  first-named  authors  inoculated  37  strains  of  typhoid  bacilli  into 
xylose  broth  and  found  that  29  of  them  produced  acid  within  twenty- 
four  hours,  while  8  of  the  strains  required  from  five  to  thirteen  days  for 
this  result.  Morishima,  of  this  "laboratory,  obtained  rapid  and  slow 
xylose  fermenters  from  a  single  strain  by  repeatedly  fishing  different 
colonies  on  plates.  An  atypical  strain  has  recently  been  described  by 
Bull  and  Pritchett9  whose  bacillus  agglutinated  in  typhoid  serum  typ- 
ically up  to  1  to  20,000,  but  which  gave  positive  indol  reactions. 

Biological  Considerations. — The  typhoid  bacillus  is  an  aerobic  and 
facultatively  anaerobic  organism  growing  well  both  in  the  presence  and 
in  the  absence  of  oxygen  when  certain  sugars  are  present,  showing  a 
slight  preference,  however,  for  well  aerated  conditions.  It  grows  most 
luxuriantly  at  temperatures  about  37.5°  C.,  but  continues  to  grow  within 
a  range  of  temperature  lying  between  15°  and  41°  C.  Its  thermal  death 
point,  according  to  Sternberg,  is  56°  C.  in  ten  minutes.  It  remains  alive 
in  artificial  cultures  for  several  months  or  even  years  if  moisture  is  sup- 
plied. In  carefully  sealed  agar  tubes  Hiss  found  the  organisms  alive 
after  thirteen  years.  In  natural  waters  it  may  remain  alive  as  long 

7  Krumwiede,  Kohn  and  Valentine,  Jour,  of  Med.  Res.,  38,  1918,  89. 

8  Morishima,  Jour,  of  Bacter.,  March,  1921. 

9  Bull  and  Pritchett,  Jour,  of  Exper.  Med.,  24,  1916,  55. 


BACILLI   OF   THE    COLON-TYPHOID-DYSENTERY   GROUP     G49 

as  thirty-six  days,  according  to  Klein.10  In  ice,  according  toJPruddeia,11 
it  may  remain  alive  for  three  months  or  over.  Against  the  ordinary 
disinfectants,  the  typhoid  bacillus  is  comparatively  more  resistant 
than  some  other  vegetative  forms.  It  is  killed,  however,  by  1  :  500 
bichlorid  or  5  per  cent  carbolic  acid  within  five  minutes. 

Pathogenicity. — In  animals,  some  early  investigators  to  the  con- 
trary, typhoidal  infection  does  not  occur  spontaneously  and  artificial 
inoculation  with  the  typhoid  bacillus  does  not  produce  a  disease  anal- 
ogous to  typhoid  fever  in  the  human  being.  Frankel 12  was  able  to 
produce  intestinal  lesions  in  guinea-pigs  by  injection  of  the  bacilli  into 
the  duodenum,  and  recovered  the  bacteria  from  the  spleens  of  the  animals 
after  death,  but  the  disease  produced  was  in  no  other  respect  analogous 
to  typhoid  fever  in  the  human  being.  It  is  probable  that  typhoid  bacilli 
injected  into  animals  do  not  multiply  extensively  and  that  most  of  the 
symptoms  produced  are  due  to  the  poisons  liberated  from  the  dead 
bacteria.  In  corroboration  of  this  view  is  the  observation  that  inocu- 
lation with  dead  cultures  is  followed  by  essentially  the  same  train  of 
symptoms  as  inoculation  with  live  cultures.13  The  injection  of  large 
doses  into  rabbits  or  guinea-pigs  intravenously  or  intraperitoneally  is 
usually  followed  by  a  rapid  drop  in  temperature,  often  by  respiratory 
embarassment  and  diarrhea.  Occasionally  blood  may  be  present  in 
the  stools.  According  to  the  size  of  the  dose  or  the  weight  of  the 
animal,  death  may  ensue  within  a  few  hours,  or,  with  progressive 
emaciation,  after  a  number  of  days,  or  the  animal  may  gradually  recover. 

Welch  and  Blachstein  14  have  shown  that  typhoid  bacilli  injected 
into  the  ear  vein  of  a  rabbit  appear  in  the  bile  and  may  persist  in  the 
gall-bladder  for  weeks.  Doerr,15  Koch,16  Morgan,17  and  more  recently 
Johnston 18  have  all  confirmed  this,  the  last  named  showing  that 
the  typhoid  bacillus  could  not  only  remain  latent  for  a  long  time 
in  the  gall-bladder  of  rabbits,  but  would  appear  in  the  blood 
stream  with  considerable  regularity  after  the  seventh  or  ninth  day, 
and  persist  in  the  gall-bladder  for  as  long  as  one  hundred  and 

10  Klein,  Med.  Officers'  Report,  Local  Govern.  Bd.,  London,  1894. 

11  Prudden,  Med.  Rec.,  1887. 

12  Frankel,  Cent,  f .  klin.  Med.,  10,  1886. 

13  Petruschky,  Zeit.  f .  Hyg.,  xii,  1892. 

14  Welch  and  Blachstein,  Bull.  Johns  Hop.  Hosp.,  ii,  1891. 

15  Doerr,  Centralbl.  f.  Bakt.,  1905. 

16  Koch,  Zeitschr.  f.  Hyg.,  1909. 

17  Morgan,  Jour,  of  Hyg.,  1911. 

18  Johnston,  Jour,  of  Med.  Res.,  xxvii,  1912.  • 


650  PATHOGENIC   MICROORGANISMS 

twenty-five  days.  Gay  and  Claypole19  have  been  able  to  produce 
the  carrier  state  in  rabbits  with  great  regularity  by  growing  the  typhoid 
cultures  used  for  inoculation  upon  agar  containing  10  per  cent  defib- 
rinated  rabbit's  blood.  Such  cultures  are  not  as  readily  agglutinated 
by  immune  serum  as  are  those  grown  on  plain  agar,  and  it  may  well  be 
that  they  have  acquired  a  certain  degree  of  resistance  to  the  serum 
antibodies  which  renders  them  more  competent  to  survive  in  the  body 
of  the  rabbit.  Gay  has  used  rabbits  inoculated  with  such  cultures  for 
the  determination  of  the  efficacy  of  his  sensitized  vaccines. 

Typical  typhoid  fever  simulating  the  disease  in  man  has  not  been 
produced  in  any  animals  except  in  chimpanzees,  by  Metchnikoff  and 
Besredka,20  who  produced  it  in  connection  with  their  experiments  on 
protective  vaccination.  They  produced  a  disease  almost  identical 
with  human  typhoid  by  feeding  cultures  to  chimpanzees. 

TYPHOID  FEVER  IN  MAN. — It  is  not  within  the  province  of  a  book  of 
this  kind  to  give  an  accurate  clinical  description  of  the  disease  as  it 
occurs  in  man  in  all  its  details.  The  disease  is  one  in  which  a  wide 
range  of  variation  may  occur,  and  in  which  complications  are  various 
and  manifold.  We  will,  therefore,  give  only  a  brief  account  of  the 
infection  as  it  is  relevant  to  bacteriological  work.  The  organisms  enter 
by  mouth,  with  food,  water  or  contact  with  fingers,  direct  or  indirect, 
as  described  in  the  epidemiological  section.  Subsequently,  the  organ- 
isms, which  pass  through  the  stomach  uninjured,  multiply  in  the  intes- 
tine, but  cause  no  symptoms  for  anywhere  from  seven  to  fourteen 
days.  During  this  time  they  probably  begin  to  proliferate  partly 
within  the  mucous  membrane  of  the  bowel,  although  there  is  little 
definite  knowledge  concerning  this.  The  symptoms  of  the  disease 
begin  insidiously  by  gradual  malaise,  headache,  loss  of  appetite,  sleep- 
lessness, and  during  the  first  week  of  the  actual  signs  of  infection,  the 
organisms  have  probably  penetrated  or  are  penetrating  into  the  lym- 
phatics. At  this  time  there  is  a  swelling  of  the  lymphoid  nodules  of  the 
intestine  and  Peyer's  patches,  and  there  is  a  moderate  catarrhal  inflam- 
mation of  the  mucous  membrane.  At  this  time  too  the  bacilli  enter  the 
blood  stream  and  can  be  found  in  blood  culture. 

Though  formerly  regarded  as  primarily  an  intestinal  disease,  the 
disease  is  in  truth  at  this  time  a  bacteriemia,  and  it  is  not  impossible 
that  the  intestinal  lesions  are  as  much  due  to  the  action  of  toxic  products 
which  are  excreted  in  part  through  the  intestinal  wall,  as  they  are  due 

19  Gay  and  Claypole,  Arch,  of  Inf.  Med.,  Dec.,  1913. 
'20  Metchnikoff  and  Besredka,  Ann.  de  Flnst.  Past.,  1911,  25,  193. 


BACILLI    OF    THE    COLON-TYPHOID-DYSENTERY    GROUP      651 

to  the  direct  reaction  caused  in  the  intestine  by  local  growth  of  the 
bacilli.  Secondarily,  the  bacilli  appear  and  can  be  cultivated  from 
the  spleen,  the  liver,  and  can  be  demonstrated  in  the  sinuses  and 
tissues  of  the  lymphatic  and  retroperitoneal  lymph  nodes. 

Typhoid  Bacilli  in  the  Blood  during  the  Disease. — The  investigations 
of  many  workers  have  shown  that  typhoid  bacilli  are  present  in  the 
circulating  blood  of  practically  all  patients  during  the  early  weeks  of  the 
disease.  Series  of  cases  have  been  studied  by  Castellani,21  Schottmul- 
ler22  and  many  others.  More  recently  Coleman  and  Buxton23  have 
reported  their  researches  upon  123  cases,  and  have  at  the  same  time 
analyzed  all  cases  previously  reported.  Their  analysis  of  blood  cultures 
taken  at  different  stages  in  the  disease  is  as  follows 

Of  224  cases  during  first  week,  89  per  cent  were  positive. 
Of  484  cases  during  second  week,  73  per  cent  were  positive. 
Of  268  cases  during  third  week,  60  per  cent  were  positive. 
Of  103  cases  during  fourth  week,  38  per  cent  were  positive. 
Of     58  cases  after  fourth  week,  26  per  cent  were  positive. 

The  technique  recommended  by  Coleman  and  Buxton  for  obtaining 
blood  cultures  is  that  recommended  by  Conradi,24  slightly  modified. 
The  blood  is  taken  into  flasks  each  containing  about  20  c.c.  of  the 
following  mixture : 

Ox-bile 900  c.c. 

Glycerin 100  c.c. 

Pepton 20  grams 

About  3  c.c.  of  blood  are  put  into  each  flask.  The  ox-bile,  besides  pre- 
venting coagulation,  may  possibly  neutralize  the  bactericidal  sub- 
stances present  in  the  drawn  blood.  The  flasks  are  incubated  for  eigh- 
teen to  twenty-four  hours,  at  the  end  of  which  time  streaks  are  made 
upon  plates  of  lactose-litmus-agar  and  the  organisms  identified  by 
agglutination  or  by  cultural  tests. 

European  workers  have  generally  preferred  to  make  high  dilution  of 
the  blood  in  flasks  of  bouillon,  small  quantities  of  blood,  1  to  2  c.c., 
being  mixed  with  100  to  150  c.c.  of  nutrient  broth. 

Epstein  25  has  reported  excellent  results  from  mixing  the  blood  in 

21  Castellani,  Riforma  medica,  1900. 

22  Schottmuller,  Deut.  med.  Woch.,  xxxii,  1900,  and  Zeit.  f.  Hyg.,  xxxvi,  1901. 
2!  Coleman  and  Kuxton.,  Am.  Jour,  of  Med.  Sci.,  133,  1907. 

24  Conradi,  Deut.  med.  Woch.,  xxxii,  1906. 

25  Epstein,  Proc.  N.  Y.  Path.  Soc.,  N.  S.,  vi,  1906. 


652  PATHOGENIC  MICROORGANISMS 

considerable  concentration  with  2-per-cent  glucose  agar  and  pouring 
plates. 

The  writers  in  hospital  work  have  had  equally  good  results  with  the 
bile  medium  and  with  broth  in  flasks,  rather  less  uniform  but  still  satis- 
factory results  with  the  plating  method.  In  general  it  may  be  said  that 
any  one  of  these  methods  carried  out  with  reasonable  accuracy  may  be 
satisfactorily  employed. 

Typhoid  Bacilli  in  the  Stools. — The  examination  of  the  stools  for 
typhoid  bacillus  is  performed  for  diagnostic  purposes  chiefly  in  obscure 
cases.  It  may,  furthermore,  furnish  information  of  great  hygienic 
importance.  Thus  Drigalski  and  Conradi 26  have  succeeded  in  isolating 
typhoid  bacilli  from  the  stools  of  ambulant  cases  so  mild  that  they 
were  not  clinically  suspected.  It  is  by  means  of  such  examinations 
that  the  so-called  typhoid-carriers  are  detected,  a  problem  which  is 
considered  at  length  in  the  section  dealing  with  epidemiology.  Such 
cases  have  been  known  to  harbor  the  bacilli  for  periods  as  long  as 
several  years. 

The  examination  itself  is  fraught  with  difficulties,  owing  to  the  pre- 
ponderating numbers  of  colon  bacilli  found  in  all  feces  and  the  diffi- 
culty of  isolating  the  typhoid  bacilli  from  such  mixtures. 

Reviewing  the  data  collected  by  a  number  of  investigators,  it 
seems  probable  that  the  bacilli  do  not  appear  in  the  stools,  at  least 
in  numbers  sufficient  for  recognition,  much  before  the  middle  of  the 
second  week,  or,  in  other  words,  as  pointed  out  by  Hiss,  about  the 
time  that  the  intestinal  lesions  are  well  advanced  and  ulceration  is 
occurring.  Thus  Wiltschour27  could  not  determine  their  presence 
before  the  tenth  day;  Redtenbacher,  28  in  reviewing  the  statistics,  states 
that  in  a  majority  of  cases  the  bacilli  first  appear  toward  the  end  of  the 
second  week,  and  Horton-Smith  29  could  not  find  the  bacilli  before  the 
eleventh  day.  Hiss,30  in  an  investigation  of  the  same  subject,  obtained 
the  following  results: 

First  to  tenth  day,  inclusive,  twenty-eight  cases  examined;  typhoid 
bacilli  isolated  from  three;  percentage  of  positive  cases  10.7  per  cent. 

Eleventh  to  twentieth  day,  inclusive,  forty-four  cases  examined; 
typhoid  bacilli  from  twenty-two;  percentage  of  positive  cases  50  per 
cent. 

26  Drigalski  and  Conradi,  Zeit.  f .  Hyg.,  xxxix,  1902. 

27  Wiltschour,  Cent.  f.  Bakt.,  1890. 

28  Redtenbacher,  Zeit.  f .  klin.  Med.,  xix,  1891. 

29  Horton-Smith,  Lancet,  May,  1899. 

30  Hiss,  Med.  News,  May,  1901. 


BACILLI   OF   THE   COLON-TYPHOID-DYSENTERY   GROUP     653 

Twenty-first  day  to  convalescence,  sixteen  cases  examined;  typhoid 
bacilli  isolated  from  thirteen;  percentage  of  positive  cases  81.2  per  cent. 

Stool  Examination  and  Method  of  Typhoid  Carrier  Detection. — 
Fecal  carriers  of  typhoid  bacilli  may  be  detected  by  cultural  methods 
applied  either  to  specimens  of  feces  or  to  duodenal  contents,  obtained  by 
a  tube  passed  through  the  stomach  into  the  duodenum.  The  simplest  method, 
of  course,  is  direct  examination  of  the  feces.  The  duodenal  tube,  however, 
will  probably  be  used  considerably  in  the  future,  since  in  some  cases  it  may 
be  positive  when  stool  cultures  are  negative.  As  a  matter  of  fact,  in  the  hands 
of  Garbat31  and  Nichols,32  the  duodenal  method  seems  to  have  given  more 
regular  results  than  the  stool  method. 

Stool  Examinations. — Stool  material  for  typhoid  examination  should  be 
fresh.  Preserving  stools  for  as  long  as  twelve  hours  will  diminish  positive 
findings  by  50  per  cent.  If  large  numbers  are  to  be  examined,  it  is 
a  good  plan  to  give  mild,  saline  cathartics  in  the  morning,  so  that  all 
specimens  can  be  collected  at  about  the  same  time.  It  is  best  to  collect 
specimens  by  cotton  swabs,  on  swab  sticks  thrust  into  tubes  in  which  there 
are  a  few  drops  of  salt  solution  to  prevent  drying.  We  have  found  that 
rectal  swabbing,  if  properly  carried  out,  may  be  a  valuable  method  of  collect- 
ing material.  If  it  is  absolutely  necessary  to  ship  stools  some  distance,  the 
addition  of  20  per  cent  glycerin  is  of  advantage. 

A  suspension  of  about  one  part  of  feces  to  twenty-five  parts  of  salt 
solution  is  made,  thoroughly  emulsified,  and  allowed  to  stand  to  allow  the 
large  particles  to  settle.  With  this  material,  surface  smears  are  made  with 
a  glass  rod  upon  plates  of  either  Robinson  and  Rettger's  modification  of 
Endo's  medium,  or  Krumwiede's  brilliant  green  medium,  as  described  in  the 
section  on  media.  It  is  of  advantage  to  use  the  large  plates.  A  bent  glass 
rod  is  dipped  into  the  emulsion  and  rubbed  over  the  surface  of  a  plate, 
beginning  in  the  center,  by  passing  in  concentric  circles  so  that  the  entire 
plate  is  gently  smeared.  A  second  plate  is  inoculated  in  the  same  way, 
without  redipping.  It  is  sometimes  well  to  make  similar  plates  with  a  1 :  5 
dilution  of  the  original  suspension. 

Plates  for  this  purpose  should  be  poured  and  allowed  to  dry  on  a  laboratory 
desk  for  a  few  hours  before  use,  and  should  be  kept  in  the  dark  if  Endo's 
medium  is  used.  Great  care  in  the  accurate  production  and  testing  out  of 
the  media,  should  be  taken  as  indicated  in  the  section  describing  these  media. 
The  plates  should  either  be  inverted  in  the  incubator,  or  else  earthen-ware 
covers  should  be  used.  Large  pieces  of  blotting  paper  inserted  under  the 
lid  serve  the  same  purpose. 

31  Garbat,  The  Typhoid  Carrier  Problem,  Monographs  of  the  Rock.  Inst.,  in  press. 

32  Nichols    H.   J.,   Jour.   Exp.    Med.,  xxiv,    1916,   497;    Jour,   of  A.   M.   A., 
Ixviii,  1917,  958. 


654  PATHOGENIC   MICROORGANISMS 

After  eighteen  hours  growth,  the  plates  should  be  examined  for  typical 
colonies.  Suspicious  colonies  should  be  immediately  inoculated  upon  tubes 
of  Russell  double-sugar  medium.  Slide  agglutinations  against  1 :100  dilution 
of  a  high  titer  stock  typhoid  antiserum  should  be  made  for  preliminary 
identification  from  suspicious  colonies,  of  course  together  with  morphological 
determination  by  smear  and  stain. 

Much  information  can  be  obtained  after  twelve  more  hours,  by  observa- 
tions of  the  growth  in  the  Russell  double-sugar  medium.  From  this  tube, 
then,  the  growth  can  be  emulsified  in  salt  solution,  and  macroscopic  agglu- 
tinations set  up.  This  usually  is  sufficient  to  identify  the  organism,  but  it  is 
always  well  to  set  up  a  few  sugar  fermentation  tubes. 

Duodenal  examinations  are  made  by  means  of  the  Einhorn  duodenal  tube, 
which  is  sterilized  by  boiling,  and  given  to  the  patient  the  evening  before 
the  examination  is  to  be  made,  about  three  hours  after  the  last  meal.  We 
take  our  description  chiefly  from  Garbat  who  has  had  considerable  experience 
with  this  method.  The  patient,  properly  instructed,  swallows  the  tube  without 
gagging,  with  ease,  and  retains  it  throughout  the  night.  On  the  following 
morning  it  has  usually  passed  into  the  duodenum,  and  bile  can  be  aspirated 
with  a  sterile  20  c.c.  Luer  syringe.  Suction  must  usually  be  exerted,  and 
Garbat  recommends  a  well  fitting  syringe  because  such  suction  must  often 
be  strong.  In  about  5  per  cent  of  Garbat's  cases  the  tube  remained  in  the 
stomach  and  more  difficulty  was  experienced  with  the  test.  When  there  is 
difficulty  in  obtaining  sufficient  bile,  the  patient  is  made  to  sit  up  in  bed 
with  His  head  bent  forward,  pressing  upward  on  the  abdomen  with  the  palms 
of  his  hands.  Sometimes  the  flow  of  bile  can  be  stimulated  by  a  cold  drink. 
The  bile  is  handled  bacteriologically  on  Endo  plates. 

Typhoid  Bacilli  in  the  Urine. — Careful  investigation  has  revealed 
typhoid  bacilli  in  the  urine  in  about  25  per  cent  of  all  patients.  Neu- 
mann 33  discovered  the  bacilli  in  eleven  out  of  forty-six  and  Karlinsky  34 
in  twenty-one  out  of  forty-four  cases.  Investigations  by  Petruschy,35 
Richardson,36  Horton-Smith,37  Hiss,38  and  others  have  confirmed  these 
results.  In  general  the  bacilli  have  not  been  found  before  the  fifteenth 
day  of  the  disease,  and  examination  of  the  urine,  therefore,  can  be  of 
little  early  diagnostic  value.  A  series  of  seventy-five  cases  examined 
by  Hiss  before  the  fourteenth  day  of  the  disease  did  not  once  reveal 
typhoid  bacilli  in  the  urine.  On  the  other  hand,  they  have  been  found 

33  Neumann,  Berl.  klin.  Woch.,  xxvii,  1890. 

34  Karlinsky,  Prag.  med.  Woch.,  xv,  1890. 

35  Petruschy,  Cent,  f .  Hyg.,  xxiii,  1898. 

36  Richardson,  Jour.  Exp.  Med.,  3,  1898. 

37  Horton-Smith,  Lancet,  May,  1899. 
*Hi88,  Med.  News,  May,  1901. 


BACILLI    OF    THE    COLON-TYPHOID-DYSENTERY    GROUP    655 

to  be  present  for  weeks,  months,  and,  in  isolated  cases,  for  years  after 
convalescence,  the  examination  thus  having  much  hygienic  importance. 
They  are  probably  present  in  about  12  per  cent  of  cases  during  the  early 
days  of  convalescence.  In  most  of  these,  albumin  is  present  in  the 
urine  in  considerable  quantities.  The  bacilli  usually  appear  and  dis- 
appear with  the  albuminuria. 

An  obstinate  cystitis  caused  by  typhoid  bacilli  may  follow  in  the 
path  of  typhoid  fever.  Such  cases  have  been  reported  by  Blumer,39 
Richardson,40  and  others.  Suppurative  processes  in  the  kidneys  are 
less  frequent.  It  is  noteworthy,  also,  that  in  the  course  of,  and  fol- 
lowing, typhoid  fever  Bacillus  coli  is  often  present  in  the  urine.  This 
may  obstinately  persist  for  considerable  periods  after  convalescence. 

For  examination  of  the  urine  for  typhoid  bacilli,  specimens  should  be 
taken  with  aseptic  precautions  and  planted  directly  into  equal  volumes 
of  broth.  Direct  plates  on  Endo  can  also  be  made,  or  had  better  be 
made  at  the  same  time.  It  is  relatively  easy  under  such  circumstances 
to  obtain  the  organism  if  present. 

Typhoid  Bacilli  in  the  Rose  Spots. — Neufeld 41  obtained  positive 
results  in  thirteen  out  of  fourteen  cases.  According  to  his  researches 
and  those  of  Frankel,42  the  bacilli  are  localized  not  in  the  blood,  which  is 
taken  when  the  rose  spots  are  incised,  but  are  crowded  in  large  numbers 
within  the  lymph  spaces. 

Typhoid  Bacilli  in  the  Sputum. — In  rare  cases  typhoid  bacilli  have 
been  found  in  the  sputum  of  cases  complicated  by  bronchitis,  broncho- 
pneumonia,  and  pleurisy.  Such  cases  have  been  reported  by  Chante- 
messe  and  Widal,43  Frankel,44  and  a  number  of  others.  Empyemia, 
when  it  occurs  in  connection  with  such  cases,  is  usually  accompanied  by  a 
mixed  infection.  From  a  hygienic  point  of  view  the  spread  of  typhoid 
fever  by  means  of  the  sputum  is  probably  of  rare  occurrence. 

Suppurative  Lesions  Due  to  Typhoid  Bacillus. — In  the  course  of 
typhoid  convalescence  or  during  the  latter  weeks  of  the  disease,  sup- 
purative  lesions  may  occur  in  various  parts  of  the  body.  The  most 
frequent  localization  of  these  is  in  the  periosteum,  especially  on  the  long 
bones,  and  in  the  joints.  A  considerable  number  of  such  lesions  have 


39  Blumer,  Johns  Hopk.  Hosp.  Rep.,  5,  1895. 

40  Richardson,  loc.  oit. 

41  Neufeld,  Zeit.  f .  Hyg.,  xxx,  1899. 

ri  Frankel.  Zeit.  f.  Hy^.,  xxxiv,  1909. 

4:i  ('hantemesse  and  Widiil,  Arch.  d(>  physiol.  norm,  ct  path.,  1887. 

44  Frankel,  Dent.  med.  Woch.,  xv  and  xvi,  1899. 


656  PATHOGENIC   MICROORGANISMS 

been  described  by  Welch,  Richardson,  *  and  others.  They  usually  take 
the  form  of  periosteal  abscesses,  often  located  upon  the  tibia,  occurring 
either  late  in  the  disease  or  months  after  convalescence,  and  are  char- 
acterized by  very  severe  pain.  Osteomyelitis  rnay  also  occur,  but  is 
comparatively  rare.  Subcutaneous  abscesses  and  deep  abscesses  in  the 
muscles,  due  to  this  bacillus,  have  been  described  by  Pratt.45  Synovitis 
may  also  occur. 

Meningitis,  due  to  the  typhoid  bacillus,  occurs  occasionally,  usually 
during  convalescence  from  typhoid  fever.  A  case  of  primary  typhoid 
meningitis  has  been  reported  by  Farnet.46 

Peritoneal  abscesses,  due  to  the  typhoid  bacillus,  have  been  reported. 
The  writer  47  has  reported  a  case  in  which  typhoid  bacilli  were  found  free 
in  the  peritoneal  cavity  during  typhoid  fever  without  perforation  of  the 
gut. 

Isolated  instances  of  typhoid  bacilli  in  abscesses  of  the  thyroid  and 
parotid  glands  and  in  brain  abscesses  have  been  observed. 

Typhoid  Fever  without  Intestinal  Lesions, — A  number  of  cases  have 
been  reported  in  which  typhoid  bacilli  have  been  isolated  from  the 
organs  after  death  or  from  the  secretions  during  life  of  patients  in  whom 
the  characteristic  lesions  of  typhoid  fever  have  been  lacking.  Most 
of  these  cases  must  be  regarded  as  true  typhoid  septicemias.  In  some 
cases  the  bacilli  were  isolated  from  the  spleen,  liver,  or  kidneys;  in 
others,  from  the  urine  or  the  gall-bladder.  In  a  case  observed  by 
Zinsser  the  bacilli  were  isolated  from  an  infarct  of  the  kidney  removed 
by  operation.  In  this  case  the  clinical  course  of  the  disease  had  pointed 
only  toward  the  existence  of  an  indefinite  fever  accompanied  by  symp- 
toms referable  to  the  kidneys.  The  Widal  test,  however,  was  positive. 
A  summary  of  such  cases,  together  with  several  personally  observed, 
has  been  given  by  Flexner.48 

Poisons  of  the  Typhoid  Bacillus. — Investigation  of  the  toxic 
products  of  the  typhoid  bacillus  has  occupied  the  attention  of  a  large 
number  of  workers.  The  first  to  do  experimental  work  upon  the  sub- 
ject was  Brieger49  soon  after  the  discovery  and  cultivation  of  the 
bacillus.  That  toxic  substances  can  be  obtained  from  typhoid  cultures 
is  beyond  question.  There  is,  however,  a  definite  difference  of  opinion 

*  Richardson,  Jour.  Boston  Soc.  Med.  Sci.,  5,  1900. 
«  Pratt,  Jour.  Boston  Soc.  Med.  Sci.,  3,  1899. 

46  Farnet,  Bull,  de  la  soc.  med.  des  hop.  dc  P.,  3,  1891. 

47  Zinsser,  Proc.  N.  Y.  Path.  Soc.,  1907. 

48  Flexner,  Johns  Hopk.  Rep.,  5,  1896. 

49  Brieger,  Deut.  med.  Woch.,  xxvii,  1902. 


BACILLI   OF   THE    COLON-TYPHOID-DYSENTERY    GROUP     657 

as  to  whether  these  poisons  are  so-called  endotoxins  only,  or  whether 
they  are  in  part  composed  of  soluble  toxins  comparable  to  those  of 
diphtheria  and  tetanus,  following  the  injection  of  which  antitoxic  sub- 
stances may  be  formed. 

The  evidence  so  far  seems  to  bear  out  the  original  contention  of 
Pfeiffer,50  who  first  advanced  the  opinion  that  the  poisonous  substances 
are  products  of  the  bacterial  body  set  free  by  destruction  of  the  bacteria 
by  the  lytic  substances  of  the  invaded  animal  or  human  being.  These 
poisons,  when  injected  into  animals  for  purposes  of  immunization,  in 
Pfeiffer's  experiments,  did  not  incite  the  production  of  neutralizing  or 
antitoxic  bodies,  but  of  bactericidal  and  lytic  substances.  That  these 
endotoxins  constitute  by  far  the  greater  part  of  the  toxic  products  of 
the  typhoid  bacillus  can  be  easily  demonstrated  in  the  laboratory,  by 
the  simple  experiment  of  filtering  a  young  typhoid  culture  (eight  or 
nine  days  old)  and  injecting  into  separate  animals  the  residue  of  bacilli 
and  the  clear  filtrate  respectively.  In  such  an  experiment  there  will  be 
little  question  as  to  the  overwhelmingly  greater  toxicity  of  the  bacillary 
bodies  as  compared  with  that  of  the  culture  filtrate.  On  the  other 
hand,  if  such  cultures,  especially  in  alkaline  media,  are  allowed  to 
stand  for  several  months  and  the  bacilli  thus  thoroughly  extracted  by 
the  broth,  the  toxicity  of  the  filtrate  is  found  to  be  greatly  increased. 

Nevertheless,  more  recent  experiments  by  Besredka,51  Macfadyen,52 
Kraus  and  Stenitzer,53  and  others  have  tended  to  show  that,  together 
with  such  endotoxic  substances,  typhoid  bacilli  may  produce  a  true  toxin 
which  is  not  only  obtainable  by  proper  methods  from  comparatively 
young  typhoid  cultures,  but  which  fulfills  the  necessary  requirement 
of  this  class  of  poisons  by  producing  in  treated  animals  a  true  antitoxic 
neutralizing  body. 

The  typhoid  endotoxins  may  be  obtained  by  a  variety  of  methods. 
Hahn  54  has  obtained  what  he  calls  "  typhoplasmin  "  by  subjecting  them 
to  a  pressure  of  about  four  hundred  atmospheres  in  a  Buchner  press. 
The  cell  juices  so  obtained  are  cleared  by  filtration.  Macfadyen  has 
obtained  typhoid  endotoxins  by  triturating  the  bacilli  after  freezing 

50  Pfeiffer,  Deut.  med.  Woch..  xlviii,  1894;    Pfeiffer  und  Kolle,  Zeit.  f.  Hyg.,  xxi, 
1896. 

51  Besredka,  Ann.  de  1'inst.  Pasteur,  1895,  1896. 

52  Macfadyen  and  Rowland,  Cent.  f.  Bakt.,  I,  xxx,  1901;    Macfadyen,  Cent.  f. 
Bakt.,  I,  1906. 

53  Kraus  und  Stenitzer,  Quoted  from  "Handb.  d.  Tech.,"  etc.,  1,  Fischer,  Jena, 
1907. 

i4  Hahn,  Munch,  med.  Woch  ,  xxiii,  190G. 


658  PATHOGENIC   MICROORGANISMS 

them  with  liquid  air  and  extracting  in  1  :  1000  potassium  hydrate. 
Besredka  obtained  toxic  substances  by  emulsifying  agar  cultures  of 
bacilli  in  salt  solution,  sterilizing  them  by  heating  to  60°  C.  for  about 
one  hour,  and  drying  in  vacuo.  The  dried  bacillary  mass  was  then 
ground  in  a  mortar  and  washed  in  sterile  salt  solution  which  was  again 
heated  to  60°  C.  for  two  hours.  The  remnants  of  the  bacterial  bodies 
settle  out  and  the  slightly  turbid  supernatant  fluid  contains  the  toxic 
substances. 

Vaughan  55  has  obtained  poisons  from  typhoid  bacilli  by  extracting 
at  78°  C.  with  a  2-per-cent  solution  of  sodium  hydrate  in  absolute 
alcohol.  In  this  way  he  claims  to  separate  by  hydrolysis  a  poisonous 
and  a  non-poisonous  fraction.  He  claims,  moreover,  that  this  poison- 
ous fraction  is  similar  to  the  poisons  obtained  in  the  same  way  from 
Bacillus  coli  and  the  tubercle  bacillus,  and  other  protein  substances, 
believing  that  the  specific  nature  of  such  proteids  depends  upon  the 
non-toxic  fraction. 

A  simple  method  of  obtaining  toxins  from  typhoid  bacilli  is  carried 
out  by  cultivating  the  microorganisms  in  meat-infusion  broth,  rendered 
alkaline  with  sodium  hydrate  to  the  extent  of  about  1  per  cent.  The 
cultures  are  allowed  to  grow  for  two  or  three  weeks  and  then  sterilized 
by  heating  to  60°  C.  for  one  hour,  and  allowed  to  stand  for  three  or 
four  weeks  at  room  temperature.  At  the  end  of  this  time  the  cultures 
may  be  filtered  through  a  Berkefeld  or  Pasteur-Chamberland  filter 
and  will  be  found  to  contain  strong  toxic  substances. 

The  accounts  concerning  the  thermostability  of  the  various  toxins 
obtained  are  considerably  at  variance.  In  general,  corresponding  with 
other  endotoxins,  observers  agree  in  considering  them  moderately 
resistant  to  heat,  rarely  being  destroyed  at  temperatures  below  70°  C. 
We  have  ourselves  often  boiled  typhoid  suspensions  without  destroying 
their  toxicity  for  guinea  pigs. 

Intravenous  inoculation  of  rabbits  with  typhoid  endotoxins,  if  in 
sufficient  quantity,  produces,  usually  within  a  few  hours,  a  very  marked 
drop  in  temperature,  diarrhea,  respiratory  embarrassment,  and  death. 
If  given  in  smaller  doses  or  by  other  methods  of  inoculation — sub- 
cutaneous or  intraperitoneal — rabbits  are  rendered  extremely  ill,  with  a 
primary  drop  in  temperature,  but  may  live  for  a  week  or  ten  days 
and  die  with  marked  progressive  emaciation,  or  may  survive.  Guinea- 
pigs  and  mice  are  susceptible  to  the  endotoxins,  though  somewhat 
less  so  than  rabbits. 

In  unpublished  experiments  we  have  perfused  the  isolated  guinea 
pig  heart  with  typhoid  extracts  for  prolonged  periods  without  killing  it, 


BACILLI   OF    THE   COLON-TYPHOID-DYSENTERY    GROUP      G59 

showing  that  the  poison  does  not  act  upon  the  normal  heart  muscle 
directly. 

Zinsser,  Parker  and  Kuttner  55  have  recently  shown  that  in  broth 
cultures  of  the  typhoid  bacillus  as  young  as  five  to  six  hours,  a  mildly 
toxic  substance  is  formed  which  can  be  recovered  in  filtrates,  and  which, 
injected  into  rabbits  intravenously,  gives  rise  to  definite  symptoms, 
after  an  incubation  time  of  an  hour  or  more.  This  substance  is  not 
specific  in  that  it  is  formed  by  many  other  different  bacteria  similarly 
grown,  and  is  not  antigenic  in  all  probability.  It  can  also  be  obtained 
by  washing  young  agar  growths  repeatedly  in  salt  solution,  and  filter- 
ing. Whether  or  not  this  substance  plays  a  part  in  the  disease  cannot 
be  stated  at  the  present  time. 

IMMUNITY    AND    ANTIBODIES 

Animals,  may  be  actively  immunized  by  the  injection  of  typhoid 
bacilli  in  gradually  increasing  doses.  In  actual  practice,  this  is  best 
accomplished  by  beginning  with  an  injection  of  about  1  c.c.  of  broth 
culture  heated  for  ten  minutes  at  60°  in  order  to  kill  the  bacilli.  After 
five  or  six  days,  a  second  injection  of  a  larger  dose  of  dead  bacilli  is 
administered;  at  similar  intervals,  gradually  increasing  doses  of  dead 
bacilli  are  given  and  finally  considerable  quantities  of  a  living  and  fully 
virulent  culture  may  be  injected  without  serious  consequences  to  the 
animal.  While  this  method  is  convenient  and  usually  successful,  it 
is  also  possible  to  obtain  satisfactory  immunization  by  beginning  with 
very  small  doses  of  living  microorganisms,  according  to  the  early 
method  of  Chantemesse  and  Widal,56  and  others. 

Such  active  immunization,  successfully  carried  out  upon  rabbits  and 
guinea-pigs,  within  a  short  time  after  the  discovery  of  the  typhoid  bacil- 
lus, was  believed  to  depend  upon  the  development  of  antitoxic  sub- 
stances in  immunized  animals.  This  point  of  view,  however,  was  not 
long  tenable,  and  was  definitely  disproven  by  the  investigations  of 
Pfeiffer  and  Kolle  57  in  1896.  These  investigators,  as  well  as  a  large 
number  of  others  working  subsequently,  have  shown  that  there  are 
present  in  the  blood  serum  of  typhoid-immune  animals  and  human 
beings,  bacteriolytic,  bactericidal,  and  agglutinating  substances,  and 
to  a  lesser  extent,  precipitating  and  opsonic  bodies  but  no  true  anti- 
toxins. 

55  Zinsser,  Parker  and  Kuttner,  Proc.  of  the  Soc.  Exper.  Med.  and  Biol.,  Meeting, 
Nov.,  1920. 

56  Chantemesse  and  Widal,  Ann.  de  Finst.  Pasteur,  1892. 

57  Pfeiffer  und  Kolle,  Zeit.  f.  Hyg.,  xxi,  1896. 


660  PATHOGENIC   MICROORGANISMS 

One  attack  of  typhoid  fever  protects  against  subsequent  infection. 
Accurate  statistics  upon  the  -matter  have  been  difficult  to  obtain, 
however,  because  histories  of  the  disease  are  apt  to  be  indefinite,  and 
until  recently,  no  proper  differentiation  was  made  between  true  typhoid 
fever  and  the  paratyphoid  group.  However,  taking  into  consideration 
these  possibilities  of  error,  the  estimations  made  by  various  clinicians 
who  have  studied  the  subject,  indicate  that  a  second  attack  of  typhoid 
fever  occurs  in  not  more  than  from  0.7  to  4  per  cent  of  all  cases.  Two 
to  3  per  cent  represents  a  fair  average  of  all  estimates  made.  When 
typhoid  fever  does  occur  for  the  second  time,  it  is  usually  of  a  milder 
type  than  the  first  attack,  though,  according  to  V.  Vaughan,  Jr.,  this 
is  not  always  the  case. 

Circulating  antibodies  disappear  from  the  typhoid  convalescent 
usually  within  the  first  seven  months  after  recovery.  Permanent 
immunity  cannot,  therefore,  be  explained  upon  the  basis  of  serum  anti- 
bodies. The  ultimate  cause  for  permanent  immunity,  in  all  diseases 
in  which  it  occurs,  must  be  regarded  as  depending  upon  the  physiological 
unit,  namely,  the  tissue  cell.  It  is  likely  that  individuals  who  have 
passed  through  an  infection  of  this  nature,  thereafter  retain  a  capacity 
to  react  more  rapidly  and  effectively  to  small  quantities  of  introduced 
antigen.  A  case  in  point  is  the  well-known  experiment  of  Wassermann, 
who  immunized  a  number  of  rabbits  to  typhoid  bacilli  until  a  highly 
agglutinin  titer  was  produced.  He  kept  these  rabbits  until  their  blood 
had  returned  to  normal  and  no  agglutinins  could  be  found.  Subse- 
quently he  reinoculated  them  with  typhoid  bacilli,  at  the  same  time 
giving  a  number  of  normal  control  rabbits  similar  injections.  The 
previously  treated  rabbits  responded  with  a  rapid  and  powerful  anti- 
body production  in  contrast  to  the  slower  antibody  curve  of  those  that 
had  received  the  typhoid  antigen  for  the  first  time.  Recent  observa- 
tions by  Moon  on  revaccination  of  previously  vaccinated  people,  have 
given  analogous  results;  and  it  appears  from  this  that  a  person,  once 
immunized,  is  capable  of  reacting  with  much  greater  promptness  than 
a  normal  individual.  Our  own  idea  would  be  somewhat  as  follows: 

When  the  typhoid  bacillus  enters  the  bowel  of  the  infected  subject 
it  begins  to  proliferate  and  gradually  enters  into  the  lymphatic  system. 
As  a  consequence,  a  small  amount  of  antigen  is  gradually  introduced 
into  the  circulation,  reaches  the  cells  and  stimulates  antibody  produc- 
tion. In  the  normal  individual  this  reaction  is  a  slow  one,  and  the 
typhoid  bacilli  multiply  with  a  speed  disproportionate  to  the  appearance 
of  antibodies.  In  the  previously  immunized  individual  or  in  the  person 
who  has  had  the  disease,  the  first  absorption  of  small  amounts  of  antigen 


BACILLI   OF   THE    COLON-TYPHOID-DYSENTERY   GROUP     661 

is  followed  by  a  tissue  reaction  (a  part  of  which  is  evident  as  antibody 
production)  so  rapid  that  the  protective  processes  are  developed  with 
sufficient  potency  to  check  the  infection  before  it  has  reached  a  phase  at 
which  symptoms  become  apparent. 

Bactericidal  and  Bacteriolytic  Substances. — The  bacteriolytic  sub- 
stances in  typhoid-immune  serum  may  be  demonstrated  either  by  the 
intraperitoneal  technique  of  Pfeiffer  or  in  vitro.  In  the  former  experi- 
ment a  small  quantity  of  a  fresh  culture  of  typhoid  bacilli  is  mixed 
with  the  diluted  immune  serum  and  the  emulsion  injected  into  the 
peritoneal  cavity  of  a  guinea-pig.  Removal  of  peritoneal  exudate  with 
a  capillary  pipette  and  examination  in  the  hanging  drop  will  reveal, 
within  a  short  time,  a  swelling  and  granulation  of  the  bacteria — the 
so-called  Pfeiffer  phenomenon.  The  test  in  vitro,  as  recommended  by 
Stern  and  Korte,58  may  be  carried  out  by  adding  definite  quantities  of 
a  fresh  agar  culture  of  typhoid  bacilli  to  progressively  increasing  dilu- 
tions of  inactivated  immune  serum  together  with  definite  quantities 
of  complement  in  the  form  of  fresh  normal  rabbit  or  guinea-pig  serum. 
At  the  end  of  several  hours'  incubation  at  37.5°  C.  definite  quantities 
of  the  fluid  from  the  various  tubes  are  inoculated  into  melted  agar 
and  plates  are  poured  to  determine  the  bactericidal  action.  Careful 
colony  counting  in  these  plates  and  comparison  with  proper  controls 
will  not  only  definitely  demonstrate  the  presence  of  bactericidal  sub- 
stances in  the  immune  serum,  but  will  furnish  a  reasonably  accurate 
quantitative  estimation.  (For  technic  of  these  tests  see  page  307.) 

Although  normal  human  serum  contains  in  small  quantity  sub- 
stances bactericidal  to  typhoid  bacilli,  moderate  dilution,  1  :  10  or 
1  :  20,  of  such  serum  will  usually  suffice  to  eliminate  any  appreciable 
bactericidal  action.  The  bactericidal  powers  of  immune  serum,  on  the 
other  hand,  are  often  active,  according  to  Stern  and  Korte,  in  dilutions 
of  over  1  :  4000.  The  specificity  of  such  reactions  gives  them  a  con- 
siderable degree  of  practical  value,  both  in  the  biological  identification 
of  a  suspected  typhoid  bacillus  in  known  serum  and  in  the  diagnosis 
of  typhoid  fever  in  the  human  patient  by  the  action  of  the  patient's 
serum  on  known  typhoid  bacilli.  In  the  publication  of  Stern  and 
Korte,  quoted  above,  it  was  found  that  typhoid  patients  during  the 
second  week  often  possess  a  bactericidal  power  exceeding  1  :  1000, 
whereas  the  blood  of  normal  human  beings  was  rarely  active  in 
dilutions  exceeding  1  :  50  or  1:  100.  While  scientifically  accurate 
the  practical  application  of  bactericidal  determinations  for  diagnosis 

58  Stem  und  Korte,  Berl.  klin.  Woch.,  x.,  1904. 


662  PATHOGENIC   MICROORGANISMS 

presents  considerable  technical  difficulties  and  gives  way  to  the  no 
less  accurate  and  much  simpler  method  of  agglutination. 

Agglutinins. — Agglutinins  are  formed  in  animals  and  man  inoculated 
with  typhoid  bacilli,  and  in  the  course  of  typhoid  fever.  It  was,  in  fact, 
while  studying  the  typhoid  bacillus  that  the  agglutinins  were  first  dis- 
covered by  Gruber  and  Durham. 

In  animals,  by  careful  immunization,  specific  typhoid  agglutinins 
may  easily  be  produced  in  sufficient  quantity  to  be  active  in  dilution 
of  1  :  10,000,  and  occasionally  even  1  :  50,000  or  over.  In  the  blood 
of  typhoid  patients,  the  agglutinins  may  often  be  found  in  dilutions. of 
1  :  100  and  over.  It  is  interesting  to  note  that  irrespective  of  the 
agglutinin  contents  of  any  given  serum,  there  may  occasionally  be  noted 
differences  in  the  agglutinability  of  various  typhoid  cultures,  a  point 
which  is  practically  important  in  the  choice  of  a  typhoid  culture  for 
routine  diagnosis  work.  Weeny  59  has  called  attention  to  the  fact  that 
bacilli  which  do  not  readily  agglutinate  when  directly  cultivated  from 
the  body,  may  often  be  rendered  more  sensitive  to  this  reaction  by  sev- 
eral generations  of  cultivation  upon  artificial  media.  Walker  has 
noted  60  a  loss  of  agglutinability  if  the  bacilli  are  cultivated  in  immune 
serum.  A  similar  alteration  in  the  agglutinability  of  typhoid  bacilli 
was  noted  by  Eisenberg  and  Volk61  when  they  subjected  the  micro- 
organism to  moderate  heat  or  to  weak  acids  such  as  ^  HC1. 

Morishima  62  has  recently  studied  the  same  phenomenon,  and  has 
confirmed  the  observations  of  Eisenberg  and  Volk,63  and  others.  He 
has  also  shown,  however,  that  if  organisms  are  cultivated  in  anti-serum 
for  a  sufficiently  long  time,  their  preliminary  inagglutinability  will 
eventually,  after  twenty  to  seventy-five  days,  revert  to  almost  normal 
agglutinability . 

Practical  application  of  agglutination  to  bacteriological  work  is 
found  in  the  identification  of  suspected  typhoid  bacilli,  and  in  the 
diagnosis  of  typhoid  fever. 

When  it  is  desired  to  determine  whether  or  not  a  given  bacillus 
is  a  typhoid  bacillus,  mixtures  may  be  made  of  young  broth  cultures, 
or  preferably  of  emulsions  of  young  agar  cultures  in  salt  solution, 
with  dilutions  of  immune  serum.  The  tests  are  made  microscopically 
in  the  hanging-drop  preparation  or,  preferably,  macroscopically  in 

69  Weeny,  Brit.  Med.  Jour.,  1889. 

60  Walker,  Jour,  of  Path,  and  Bad.,  1892;   Totxtikn,  Holt.  f.  HyR.,  xlv,  1903. 

01  Eixenberg  und  Volk,  Zeit.,  f.  Hyg.,  xlv,  190:5. 

^Morishima,  Jour,  of  Baeter.,  March,  1921. 

63  Eisenberg  and  Volk,  Erbeg.  der  Immunit.  Exper.  Ther.  Bakt.  u.  Hyg.,  1913,  73. 


BACILLI   OF   THE    COLON-TYPHOlB-DYSENTERY    GROUP     663 

small  test  tubes.  In  all  cases  it  is  desirable  first  to  determine  the 
agglutinating  power  of  the  scrum  when  tested  against  a  known 
typhoid  culture.  (For  detailed  technique,  see  chapter  on  Technique 
of  Serum  Reactions,  page  302,  282.) 

In  scientific  investigations,  specific  agglutinations  in  high  dilutions 
of  immune  serum  constitute  very  strong  proof  of  the  species  of  the  micro- 
organism and  may  often  furnish  much  information  as  to  the  biological 
relationships  between  similar  species.  It  is  found  in  immunizing  ani- 
mals with  any  given  strain  of  typhoid  bacilli,  that  there  are  formed 
the  " chief"  or  " major"  agglutinins  which  are  specific  and  active 
against  the  species  used  in  immunization,  and  the  "-group"  or  "minor" 
agglutinins,  active  also  against  closely  related  microorganisms.  The 
following  extract  from  a  table  will  serve  to  illustrate  this  point  in  the 
case  of  typhoid  and  allied  bacilli. 


Highly  Immune  Typhoid  Scrum. 

1   :  100 

1  :  250 

1   :  500 

1  :  1000 

1   :  2500 

B.  typh  
B.  paratyph.  (Schottmiiller)  ...... 
B.  enteritidis  

+ 
+ 

+ 
+ 

+ 
+ 

+ 
+ 

+ 

+ 

B.  ooli  communis  

The  sera  of  most  adult  normal  animals  and  human  beings  usually 
contain  a  small  amount  of  agglutinin  for  these  bacilli.  Immunization 
with  the  typhoid  bacillus,  while  increasing  chiefly  the  agglutinin 
for  this  bacillus  itself,  also  to  a  slighter  extent  increases  the  group 
agglutinins  for  other  closely  allied  species.  That  these  group  agglu- 
tinins are  separate  substances  and  not  merely  a  weaker  manifestation 
of  the  action  of  the  typhoid  agglutinin  itself  upon  these  other  micro- 
organisms, may  be  demonstrated  by  the  experiments  of  agglutinin 
absorption.  (See  section  on  Agglutinins,  page  288.) 

In  the  clinical  diagnosis  of  typhoid  fever,  the  phenomenon  of  agglu- 
tination was  first  utilized  by  Widal.64  This  observer  called  attention 
to  the  fact  that  during  the  last  part  of  the  first  or  the  earlier  days  of  the 
second  week  of  typhoid  fever,  as  well  as  later  in  the  disease  and  in  con- 
valescence, the  blood  serum  of  patients  would  cause  agglutination  of 
typhoid  bacilli  in  dilutions  of  1  :  10,  or  over,  whereas  the  serum,  of 
normal  individuals  usually  exerted  no  such  influence.  Upon  this  basis 

64  Widal,  Bull,  de  la  soc.  mcd.  des  hopit.,  vi,  1896;  Widal  et  Sicard,  Ann.  de 
1'inst.  Pasteur,  xi,  1897. 


664  PATHOGENIC   MICROORGANISMS 

he  recommended,  for  the  diagnosis  of  the  disease,  the  employment  of  a 
microscopic  agglutination  test  carried  out  by  the  usual  hanging-drop 
technique.  The  reaction  of  Widal  is,  at  present,  widely  depended  upon 
for  diagnostic  purposes  and  although  not  universally  successful,  owing 
to  irregularities  in  agglutinin  formation  in  some  patients  and  because  of 
differences  in  agglutinability  of  the  cultures  employed,  it  is  nevertheless 
of  much* value.  The  fact  that  the  recent  work  of  Hooker  and  of  Weiss 
has  shown  that  typhoid  bacilli  differ  in  antigenic  properties,  and  may 
on  the  basis  of  agglutination  and  agglutinin  absorption  be  divided  into  a 
number  of  groups,  is  not  of  sufficient  practical  importance  to  necessitate 
the  use  of  a  variety  of  strains  since  the  atypical  antigenic  ones  are  rela- 
tively rare.  Original  conclusions  as  to  the  dilutions  of  the  serum  which 
must  be  employed,  have,  however,  necessarily  been  modified.  Owing 
to  the  fact  that  Gruber,65  Stern,66  Frankel,67  and  a  number  of  others 
have  found  that  occasionally  normal  serum  will  give  rise  to  agglutina- 
tion of  typhoid  bacilli  in  dilutions  exceeding  1  :  10,  it  has  been  found 
necessary,  whenever  making  a  diagnostic  test,  to  make  several  dilutions, 
the  ones  most  commonly  employed  being  1  :  20,  1  :  40,  1  :  60,  and 
1  :  80.  The  wide  application  of  the  method  has  given  rise  to  the 
development  of  a  number  of  technical  procedures,  all  of  them  devised 
with  a  view  toward  simplification.  In  ordinary  hospital  work,  it  is 
most  convenient  to  keep  on  hand  upon  slant  agar,  a  stock  typhoid 
culture,  the  agglutinability  of  which  is  well  known.  From  this  stock 
culture,  fresh  inoculations  upon  neutral  bouillon  should  be  made  each 
day,  so  that  a  young  broth  culture  may  always  be  on  hand  to  furnish 
actively  motile,  evenly  distributed  bacteria.  These  bouillon  cultures 
may  be  grown  for  from  six  to  eight  hours  at  incubator  temperature  or 
for  from  twelve  to  eighteen  hours  at  room  temperature.  The  tempera- 
tures at  which  the  broth  cultures  are  kept  .must  depend,  to  a  certain 
extent,  upon  the  peculiarities  of  the  typhoid  bacillus  employed,  since 
some  strains  are  more  actively  motile  and  furnish  a  more  suitable 
emulsion  if  kept  at  a  temperature  lower  than  37.5°  C.  A  false  clumping 
in  the  broth  cultures  due  to  a  too-high  acidity  of  the  bouillon  or  a  too- 
prolonged  incubation  must  be  carefully  guarded  against.  It  is  also 
possible  to  use  for  this  test  an  emulsion  of  typhoid  bacilli  prepared  by 
rubbing  up  a  small  quantity  of  a  young  agar  culture  in  salt  solution. 
Uniformity  in  the  preparation  of  broth  cultures  or  of  emulsions  should  be 
observed,  since  the  quantitative  relationship  between  typhoid  bacilli 

65  Gruber,  Verhand.  Congr.  f.  inn.  Med.,  Wiesbaden,  1896. 

66  Stern,  Cent.  f.  inn.  Med.,  xlix,  1896. 

67  Frankel,  Deut.  med.  Woch.,  ii,  1897. 


BACILLI   OF   THE    COLON-TYPHOID-DYSENTERY    GROUP     665 

and  agglutinins  will  markedly  affect  the  completeness  or  incompleteness 
of  the  reaction.  In  high  dilutions  an  excess  of  typhoid  bacilli  may  bring 
about  complete  absorption  of  all  the  agglutinins  present,  without  agglu- 
tinating all  the  microorganisms. 

The  blood  of  the  patient  to  be  used  for  a  Widal  test  may  be  obtained 
in  a  number  of  ways.  The  most  convenient  method  is  to  bleed  the 
patient  from  the  ear  or  ringer  into  a  small  glass  capsule,  in  the  form  of 
that  used  in  obtaining  blood  for  the  opsonin  test,  or  into  a  small  centri- 
fuge tube.  About  0.5  to  1  c.c.  is  ample.  These  capsules  or  tubes, 
after  clotting  of  the  blood,  may  be  placed  in  the  centrifuge  which  in 
a  few  revolutions  will  separate  clear  serum  from  clot.  The  dilutions 
of  the  serum  are  then  made.  It  is  best  to  use  sterile  physiological  salt 
solution  as  a  diluent,  but  neutral  broth  may  be  used.  The  dilutions 
may  be  made  either  by  means  of  an  ordinary  blood-counting  pipette  or 
by  means  of  a  capillary  pipette  upon  which  a  mark  with  a  grease  pencil, 
made  about  an  inch  from  the  tip,  furnishes  a  unit  of  measure,  and  upon 
which  suction  is  made  by  means  of  a  rubber  nipple.  It  is  convenient 
to  have  at  hand  a  small  porcelain  palette  such  as  that  used  by  painters, 
in  which  the  various  cup-like  impressions  may  be  utilized  to  contain  the 
various  dilutions.  Dilutions  of  the  serum  are  made,  ranging  from  1  :  10 
to  1  :  50.  A  drop  of  each  of  these  dilutions  is  mixed  with  a  drop  of  the 
typhoid  culture  or  emulsion  upon  the  center  of  a  cover-slip  and  the  cover- 
slip  inverted  over  a  hollow  slide.  A  control  with  normal  serum  and  with 
the  same  culture  should  always  be  made  and  also  one  with  the  culture 
alone  to  exclude  the  possibility  of  spontaneous  clumping.  Mixture 
with  the  typhoid  culture,  of  course,  each  time  doubles  the  dilutions 
so  that,  for  instance,  a  drop  of  serum  dilution  1  :  10,  plus  a  drop  of 
the  typhoid  culture,  gives  the  final  dilution  of  1  :  20.  The  preparation 
may  be  examined  with  a  high  power  dry  lens  or  an  oil  immersion  lens. 
In  a  positive  reaction,  the  bacilli,  which  at  first  swim  about  actively, 
singly  or  in  short  chains,  soon  begin  to  gather  in  small  groups  and  lose 
much  of  their  activity.  Within  one-half  to  one  hour,  they  will  be 
gathered  in  dense  clumps  between  which  the  fluid  is  clear  and  free  from 
bacteria,  and  only  upon  the  edges  of  the  agglutinated  masses  may  slight 
motility  be  observed.  The  degree  of  dilution  and  the  time  of  exposure 
at  which  such  a  reaction  may  be  regarded  as  of  specific  diagnostic  value 
have  been  largely  a  matter  of  empirical  determination.  It  is  generally 
/iccepted  at  present  that  complete  agglutination  within  one  hour  in 
dilutions  from  1  :  40  to  1  :  60  is  definite  proof  of  the  existence  of  typhoid 
infection.  Exceptions,  however,  to  this  rule  may  occur.  Agglutina- 
tions of  typhoid  bacilli  in  dilutions  of  1  :  40,  and  over,  have  occasionally 


666  PATHOGENIC   MICROORGANISMS 

been  observed  in  cases  of  jaundice  and  of  tuberculosis,  and  these  condi- 
tions must  occasionally  be  considered,  though  their  importance  was 
formerly  exaggerated. 

The  method  of  making  the  Widal  test  from  a  drop  of  whole  blood 
dried  upon  a  slide,  is  not  to  be  recommended'  since  accuracy  in  dilution 
by  this  method  is  practically  impossible. 

As  stated  above,  the  agglutinin  reaction  rarely  appears  in  typhoid 
fever  before  the  beginning  of  the  second  week.  It  may  continue  during 
convalescence  for  as  long  as  six  to  eight  weeks  and  occasionally,  in  cases 
where  there  is  a  chronic  infection  of  the  gall-bladder,  a  Widal  reaction 
may  be  present  for  years  after  an  attack. 

For  very  exact  work,  even  in  clinical  cases,  the  microscopic  agglu- 
tination method  may  be  replaced  by  macroscopic  agglutination,  accord- 
ing to  the  technique  described  in  another  section  (page  303.) 

In  order  to  avoid  both  the  necessity  of  keeping  alive  typhoid  cul- 
tures for  routine  agglutination  tests  and  also  to  preclude  the  danger  of 
infection  by  the  use  of  living  culture,  Ficker68  has  recommended  typhoid 
bacilli  killed  by  formalin.  This  method  has  no  advantages  for  practical 
purposes  and  in  scientific  bacteriological  work  it  is,  of  course,  not  to 
be  considered  in  comparison  with  the  more  exact  methods. 

The  more  recently  introduced  general  practice  of  vaccination  in 
typhoid  fever  has  added  a  complicating  factor  to  diagnostic  agglutina- 
tion. Individuals  so  vaccinated  develop  agglutinins  in  consequence 
of  the  inoculations,  which  may  persist  for  six  months  or  more,  and  even 
after  they  have  disappeared  from  the  blood  stream,  various  non-typhoid 
febrile  conditions  may  induce  their  appearance  in  the  circulation  for 
reasons  not  well  understood.  In  consequence  of  this,  it  is  necessary, 
before  drawing  conclusions  concerning  the  meaning  of  a  Widal  reac- 
tion, to  be  thoroughly  informed  concerning  the  vaccination  history  of 
the  patient  and  the  time  which  has  elapsed  since  the  vaccination  was 
done.  A  certain  amount  of  reliable  information  may  be  obtained  even 
in  such  cases  by  the  study  of  the  quantitative  changes  in  the  agglutinins 
in  the  patient's  blood  by  the  comparative  method  of  Dreyer  as  in  use 
in  the  United  States  Army. 

This  method,  however,  is,  in  our  opinion,  not  sufficiently  useful  or 
simple  to  be  recommended  for  ordinary  clinical  use.  In  the  Dreyer 
method,  standardized  suspensions  of  Bacillus  typhosus  or  the  para- 
typhoid types  are  used.  To  obtain  these,  cultures  of  the  bacilli  are 
grown  for  about  two  weeks  by  daily  transplant  in  broth,  a  procedure 

68  Picker,  Berl.  klin.  Woch.,  xlvii.  1903. 


BACILLI   OF   THE   COLON-TYPHOID-DYSENTERY   GROUP     667 

which  is  supposed  to  increase  agglutinability.  After  this,  it  is  planted 
in  flasks  in  broth,  allowed  to  grow  overnight,  and  0.1  per  cent  formalin 
is  added.09  This  formalinized  culture  is  placed  in  the  refrigerator  and 
shaken  frequently  during  four  or  five  days.  It  is  then  standardized 
for  opacity  against  an  arbitrary  standard  kept  on  hand,  and  prepared 
in  the  Army  Medical  School.  Necessary  dilutions  are  made  with 
physiological  salt  solution  to  which  0.1  per  cent  formalin  has  been 
added. 

The  suspension  is  then  standardized  for  agglutinability  against  a 
known  agglutinating  immune  serum.  For  this  purpose  incubation  at 
55°  for  two  hours  is  a  method  used  for  the  final  reading.  It  is  easy, 
from  this  test,  then,  to  determine  the  agglutinability  factor  of  the  new 
suspension. 

The  example  cited  in  the  Army  Medical  School  War  Manual,  No.  6, 
is  as  follows:  If  the  dilution  of  the  solution  in  which  the  standardized 
suspension  is  agglutinated  is  1  to  6400,  while  that  of  the  new  suspension 
is  1  to  3200,  then  the  factor  of  the  new  bacterial  suspension  is  one-half. 

By  the  use  of  such  suspension  it  is  clear  that  comparative  titrations 
of  the  rise  and  fall  of  agglutinins  in  the  patient's  serum  can  be  made, 
and  information  obtained  which  may  have  considerable  importance  in 
deciding  whether  the  appearance  of  agglutinins  in  the  patient  is  due 
to  previous  vaccination,  or  is  present  in  response  to  a  fresh  infection. 

Precipitins. — The  investigations  of  Kraus,70  by  which  the  pre- 
cipitins  were  discovered,  revealed  specific  precipitating  substances, 
among  others,  also  in  typhoid  immune  sera.  Since  Kraus'  original 
investigation,  these  substances  have  been  studied  by  Norris  71  and 
others.72 

Opsonins. — A  number  of  observers  have  shown  that  opsonins  specific 
for  the  typhoid  bacillus  are  formed  in  animals  immunized  with  these 
organisms.  Opsonins  are  formed  also  in  patients  suffering  from  typhoid 
fever,  but  exact  opsonic  estimations  in  all  these  cases  are  extremely 
difficult  because  of  the  rapid  lysis  which  these  bacteria  may  undergo 
both  in  the  serum,  and  intracellularly,  after  ingestion  by  the  leucocytes. 
Klein  73  has  attempted  to  overcome  this  difficulty  by  working  with 
dilutions  of  serum,  at  the  same  time  using  comparatively  thick 
bacterial  emulsions  and  exposures  to  the  phagocytic  action  not  exceed- 

69  U.  S.  Medical  War  Manual  No.  6.     Lea  &  Febiger,  1919. 

70  Kraus,  Wien.  klin.  Woch.,  xxxii,  1897. 

71  Norris,  Jour,  of  Inf.  Dis.,  I,  3,  1904. 

72  Barker  and  Cole,  22d  Ann.  Session,  Assn.  of  Amer.  Phys.,  Wash.,  1897. 

73  Klein,  Bull.  Johns  Hopkins  Hosp.,  1907. 


668  PATHOGENIC   MICROORGANISMS 

ing  ten  minutes.  Chantemesse  74  has  claimed  that  the  opsonic  index  of 
typhoid  patients  was  increased  after  treatment  with  a  serum  obtained 
by  him  from  immunized  horses,  and  Harrison  75  has  reported  similar 
results  in  patients  treated  by  a  modification  of  Wright's  method  of 
active  immunization.  Klein  claims  to  have  demonstrated  that  in 
typhoid-immune  rabbits,  after  five  injections,  the  opsonic  contents  of 
the  blood  were  increased  to  an  equal  extent  as  the  bactericidal  sub- 
stances. He  concludes  from  this  interesting  observation  that  it  may 
well  be  that  the  opsonins  are  quite  as  important  in  typhoid  immunity 
as  are  the  latter  substances. 

For  diagnostic  purposes  in  typhoid  fever  the  estimation  of  the  opsonic 
index,  so  far,  has  not  been  proven  to  be  of  great  value. 

SANITARY   CONSIDERATIONS  IN  TYPHOID  FEVER 

Typhoid  fever  is  a  disease  which  has  been  constantly  diminishing 
in  frequency  in  civilized  countries  during  the  last  one  hundred  years, 
but  is  still  a  very  formidable  cause  of  death  rate  and  disability.  The 
morbidity  rates  and  death  rates  for  typhoid  fever  vary  considerably 
in  different  communities  according  to  the  extent  to  which  sanitary 
supervision  of  water  supplies,  garbage  and  sewage  disposal,  etc.,  have 
been  developed.  In  general,  the  United  States  has  been  considerably 
behind  most  European  communities.  In  a  table  given  by  Gay  76  a 
comparison  of  mortality  averages  per  100,000  population,  comparing 
a  group  of  over  31,000,000  people  compiled  from  the  statistics  of  the 
33  largest  European  cities,  with  21,000,000  people  representing  the  pop- 
ulations of  57  of  the  largest  American  cities,  the  European  mortality 
average  was  6.5,  and  the  American  19.59.  In  similar  compilations 
taken  by  Gay  largely  from  the  report  of  the  New  York  State  Depart- 
ment of  Health  for  1914,  it  is  shown  that  there  has  been  a  progressive 
decrease  since  1910,  running  parallel  to  increased  attention  to  water 
supplies  and  general  sanitation.  For  more  extensive  figures  on  the 
prevalence  of  typhoid  fever  the  reader  is  referred  to  the  above-mentioned 
compilation  of  Gay.  He  states  that  in  1900  there  were  about  350,000 
cases  of  typhoid  fever  in  the  United  States  as  estimated  by  Whipple,  who 
at  the  same  time  calculates  that  the  cost  to  the  community  of  these 
cases  must  have  been  approximately  $212,000,000.  Since,  as  we  shall 
see,  it  has  been  variously  shown  during  the  last  ten  years  that  typhoid 

74  Chantemesse,  14th  Internal!.  Cong,  for  Hyg.,  Berlin,  1907. 

75  Harrison,  Jour.  Royal  Army  Med.  Corps,  8,  1907. 

V6  Gay,  F.  P.,  Typhoid  Fever,  Macmillan  Company,  New  York,  1918. 


BACILLI    OF    THE    COLON-TYPHOID-DYSENTERY    GROUP     669 

fever  is  a  distinctly  preventable  disease,  amenable  perhaps  not  to  com- 
plete eradication  on  account  of  the  difficulties  of  the  carrier  problem, 
but  certainly  readily  subject  to  material  diminution,  much  of  this 
suffering  and  economic  loss  would  seem  unnecessary. 

Infection  with  typhoid  fever  always  means  that  intestinal  contents 
of  a  case  or  a  carrier  have  come  into  direct  or  indirect  contact  with 
something  ingested  by  the  patient.  Since  this  is  true,  we  may  best 
begin  the  description  of  the  circle  of  infection  from  man  to  man  by 
first  considering  the  manner  in  which  the  organism  leaves  the  body  of 
the  patient  and  the  carrier. 

In  the  patient  the  typhoid  bacillus  begins  to  accumulate  in  the  intes- 
tines during  the  later  stages  of  the  incubation  time,  and  at  this  time  will 
begin  to  appear  in  the  feces.  The  organisms  increase  in  the  intestines 
from  this  time  on,  being  distributed  in  very  considerable  numbers  after 
the  second  week,  and  decreasing  only  towards  the  end  of  the  disease, 
remaining  present,  however,  throughout  convalescence  and  sometimes, 
as  we  shall  see,  for  months  or  years  thereafter.  During  the  second  and 
third  or  later  weeks,  the  organisms  appear  in  the  urine.  It  is  generally 
stated  that  about  30  per  cent  of  typhoid  cases  will  show  the  organisms 
in  the  urine,  but  it  seems  likely  that  this  is  too  low  an  estimate.  Rau- 
bitschek,  by  precipitating  considerable  quantities  of  urine  with  ferric 
chlorate  succeeded  in  finding  the  bacilli  in  100  per  cent  of  his  cases  in 
the^  earlier  stages  of  the  disease,  and  it  is  not  at  all  Unlikely  that  in  slight 
numbers,  and  perhaps  intermittently,  they  may  appear  in  the  urine  of 
all  typhoid  cases.  Other  routes  of  distribution  from  the  patient,  such 
as  suppurations,  sputum,  etc.,  are  occasionally  mentioned,  but  may  be 
dismissed  as  of  no  practical  sanitary  importance. 

Since  the  recognized  typhoid  case  is  usually  well  guarded  from  a 
sanitary  point  of  view,  the  greater  danger  of  typhoid  infection  lies  in 
the  mild,  atypical,  unrecognized  case  and  in  the  carrier.  Atypical, 
mild  cases  will  probably  become  more  and  more  frequent  as  typhoid 
vaccination  becomes  a  more  generalized  habit.  Such  a  case  may  show 
nothing  more  than  a  very  slight  febrile  movement,  with  intestinal  dis- 
turbances and  diarrhea.  Unless  typhoid  fever  is  particularly  looked 
for  and  suspected,  many  of  these  cases  may  never  be  put  upon  typhoid 
precautions  and  the  resulting  possibilities  of  spread  are  obvious. 

More  important  from  the  sanitary  point  of  view,  under  the  con- 
ditions of  modern  community  life,  however,  is  the  typhoid  carrier. 

TYPHOID  CARRIERS. — The  great  importance  of  the  typhoid  carrier 
in  the  spread  of  the  disease  has  led  to  extensive  studies  of  the  problem 
in  many  countries  during  the  last  ten  years.  We  may  mention  par- 


G70  PATHOGENIC   MICROORGANISMS 

ticularly  the  studies  of  Conradi  and  Drigalski77  the  paper  of  Sacquepee,78 
the  summary  given  by  Kutscher  79  in  the  Second  Edition  of  the  Kolle 
and  Wassermann  Handbook,  the  summary  of  Gay  in  the  book  men- 
tioned above,  and  the  article  by  Garbat,  not  yet  published,  but  about  to 
appear  as  one  of  the  Monographs  of  the  Rockefeller  Institute.  The 
first  definite  suggestion  of  the  danger  of  typhoid  infection  emanating 
from  convalescents  long  after  the  disease  itself  had  been  cured,  came 
from  Koch.80  He  based  this  opinion  at  first  upon  purely  epidemiological 
evidence,  but  in  1904  Drigalski  81  began  to  isolate  bacilli  from  individuals 
who  were  apparently  in  complete  health.  Sacquepee 78  classifies 
typhoid  carriers  chiefly  into  convalescent  carriers  who  become  free  of 
the  bacilli  within  three  months  after  the  termination  of  their  disease, 
and  chronic  carriers  who  continue  to  harbor  the  bacilli  for  many  years, 
and  perhaps  permanently.  In  addition  to  this,  there  are  a  certain 
number  of  so-called  healthy  carriers  in  whom  no  history  of  their  ever 
having  had  the  disease  can  be  adduced. 

The  distinction  between  a  temporary  carrier  and  a  chronic  carrier 
must,  of  course,  be  to  a  certain  degree  arbitrary,  but  in  general  it  may 
be  said  that  in  most  typhoid  cases  the  organisms  disappear  from  the 
urine  and  feces  within  from  six  weeks  to  three  months  after  recovery. 
Sacquepee  classifies  as  chronic  carriers  only  those  in  which  the  organisms 
are  still  present  three  months  after  complete  recovery.  After  this 
period,  the  length  of  time  to  which  the  carrier  may  persist  is  variable, 
depending  upon  whether  or  not  chronic  lesions  are  established.  These 
will  be  discussed  below.  The  frequence  with  which  chronic  carriers 
following  typhoid  fever  occur  may  be  gathered  from  the  table  compiled 
by  Gay  and  published  in  the  book  mentioned  above. 

If  we  consider  that  the  figures  presented  in  this  table  must  neces- 
sarily represent  underestimates  because  of  the  technical  difficulties 
attending  the  discovery  of  small  numbers  of  typhoid  bacilli,  it  becomes 
apparent  that  the  number  of  potential  foci  for  infection  in  a  community 
is  enormous.  Gay  estimates,  on  a  basis  of  a  5  per  cent  minimum,  that 
we  have  7500  added  annually  to  the  carriers  present  in  the  United 
States.  On  this  basis  about  0.2  per  cent  to  0.3  per  cent  of  the  general 
population  may  be  assumed  to  be  carriers. 

According  to  the  foci  upon  which  the  carrier  state  depends,  typhoid 

77  Conradi  and  Drigalski,  Zeit.  f.  Hyg.,  34,  1902,  283. 

78  Sacquepee,  Bull,  do  I'lnst.  Past,,  8,  1910,  521  and  689. 

79  Kutscher,  Kolle  and  VVassonnann  Ilandbuch,  2d  Edition,  Fischer,  Jena,  1913. 
™Koch,  Ver.  a.  d.  Militarsanitatswesen,  H.  21,  1902. 

81  Drigalski,  Cent.  f.  Bakt,,  35,  1904,  776. 


BACILLI    OF   THE    COLON-TYPHOID-DYSENTERY    GROUP     671 

carriers  have  been  subdivided  by  a  number  of  writers  into  intestinal 
carriers,  gall-bladder  carriers,  and  liver  or  bile  duct  carriers.  We  will 
see  how  the  newer  methods  of  duodenal  tube  examination  have  made 
these  distinctions  between  carriers  possible. 

PERCENTAGES     OF     CHRONIC     TYPHOID     CARRIERS     FOUND     BY 
VARIOUS     INVESTIGATORS    IN     A     STUDY    OF    CONVALESCENT 

CASES  * 


Author. 

Date. 

Number  of 
Cases. 

Percentage 
Carriers  for 
3  Months 
and  More. 

Lentz  

1905 

400 

3  0 

Conradi  

1907 

400 

0.5 

Klinger                      .    . 

1907 

482 

1  7 

Kayser 

1907 

101 

3  5 

Semple  and  Greig  

1908 

86 

11.6 

Park  

1908 

68 

5.9 

Tsuzuki 

1910 

51 

5  8 

Bruckner  

1910 

316 

3.8 

Stokes  and  Clarke  

1916 

810 

1  85 

*  Table  taken  from  Gay,  F.  P.,  Typhoid  Fever,  MacMillan  &  Co.,  New  York, 
1918. 

By  far  the  most  common  localization  of  typhoid  bacilli  in  the  body 
of  the  carrier  is  the  gall  bladder.  In  speaking  of  the  sequela?  of  typhoid 
fever  we  have  seen  that  cholecystitis  is  almost  always  related  to  a  pre- 
ceding attack  of  typhoid  fever.  As  a  matter  of  fact  in  the  course  of 
typhoid  fever  the  organisms  are  always  present  in  the  gall  bladder. 
This  was  noted  by  Chiari  82  as  early  as  1894,  by  Pratt,83  and  by  many 
others.  Longcope  is  quoted  by  Gay  to  have  taken  bile  cultures  as  a 
routine  in  suspected  typhoid  deaths  at  the  Pennsylvania  Hospital,  and 
found  typhoid  bacilli  in  all  positive  cases.  In  the  gall  bladder  appar- 
ently the  organisms  find  a  protected  nidus  where  they  can  persist 
for  years.  If  gall  stones  are  formed  later,  typhoid  bacilli  can  often 
be  isolated  from  them.  We  ourselves  have  reported  a  case  in  which  we 
isolated  the  organisms  from  gall  stones  seventeen  years  after  the  attack 
of  typhoid. 

That  liver-duct  carriers,  however,  may  exist  independently  of  gall- 
bladder infection  has  been  shown  by  such  cases  as  the  one  cited  by 
Garbat  in  the  essay  mentioned  above.  He  speaks  of  two  patients 


82  Chiari,  Cent,  f .  Bakt.,  Orig.,  15,  1894. 

83  Pratt,  Amer.  Jour.  Med.  Sciences,  1901. 


672  PATHOGENIC   MICROORGANISMS 

who,  during  typhoid  convalescence,  manifested  gall-bladder  symptoms. 
Direct  culture  of  the  bile  by  means  of  the  duodenal  tube  method  showed 
typhoid  bacilli  in  "  A,"  but  not  in  "B."  In  both,  the  gall  bladder  was 
removed  and  a  pure  culture  of  typhoid  bacilli  obtained  from  both  gall 
bladders.  At  the  time  of  operation,  the  negative  culture  in  "B"  was 
explained  by  the  fact  that  a  large  stone  was  fixed  in  the  cystic  duct  which 
completely  occluded  the  passage.  The  bile  from  "B,"  before  operation 
had  come  directly  from  the  liver,  and  had  not  entered  the  gall  bladder 
which  was  in  this  case,  the  only  site  of  infection.  After  operation 
however,  typhoid  bacilli  completely  disappeared  from  "B,"  where  the 
bile  that  had  come  from  the  liver  had  been  found  sterile  by  the  original 
duodenal  culture,  but  in  "A,"  in  spite  of  the  complete  removal  of  the 
gall  bladder  and  cystic  duct,  repeated  duodenal  cultures  remained  posi- 
tive. Similar  cases  have  been  reported  in  the  literature,  but  none  which 
seem  quite  as  convincing  as  a  proof  for  the  existence  of  the  true  liver 
carrier  as  these  instances  reported  by  Garbat. 

The  manner  in  which  typhoid  bacilli  get  into  the  gall  bladder  has 
occupied  the  attention  of  a  number  of  investigators.  According  to 
Kiister  84  and  a  more  recent  report  by  Garbat  ascending  infection  of  the 
gall  bladder  from  the  duodenal  is  a  possibility,  though  it  is  probably 
not  the  most  common  method  of  infection.  Nichols  85  too  has  admitted 
the  possibility  of  such  a  process,  although  no  one  believes  that  this  is 
very  common.  The  fact  also  that,  according  to  Blumenthal  86  Lauben- 
heimer  87  and  others,  colon  bacilli  are  very  commonly  found  in  the  gall 
bladder,  gives  support  to  the  possibility  of  ascending  infection.  The 
opinion,  however,  that  the  bile  is  hematogeneously  infected  by  way  of 
the  hepatic  circulation  in  most  cases  is  generally  accepted. 

The  existence  of  pure  intestinal  carriers  has  been  suggested  by  Kraus 
and  others,  and  in  addition  to  the  cases  cited  by  Kraus,  there  is  one  by 
Garbat  in  which  duodenal  cultures  were  repeatedly  negative,  whereas 
the  feces  remained  positive.  Cholecystectomy  on  this  case  did  not 
relieve  the  carrier  condition.  The  intestinal  carrier  type,  according 
to  Kraus 88  may  be  associated  with  chronic  intestinal  ulcerations, 
chronic  appendicitis,  etc.,  but  is  unquestionably  extremely  rare,  a  large 
majority  of  carriers  being  due  to  actual  gall-bladder  infection. 

Chronic  urinary  carriers  are  less  common  than  chronic  feces  carriers. 

MKu  ter,  Beitr.  f.  Klin.  d.  Infkrankh,  etc.,  7,  1918,  98. 

85  Nichols,  Jour,  of  the  A.  M.  A.,  68,  1917. 

86  Blumenthal,  Arch,  f .  klin.  Med.,  88,  1907,  509. 

87  Laubenheimer,  Zeit.  f .  Hyg.,  58,  1909. 

88  Kraus,  Wien.  klin.  Woch.,  27,  1914,  1443. 


BACILLI   OF   THE   COLON-TYPHOID-DYSENTERY    GROUP     673 

Yet,  when  they  occur,  they  are  of  much  greater  danger  to  others  because 
of  the  more  indiscriminate  distribution  of  urine.  According  to  Gar- 
bat  89  about  6.8  per  cent  of  all  typhoid  cases  show  typhoid  bacilluria 
for  one  or  two  months  after  the  fever  has  disappeared.  In  such  cases 
often  the  organisms  are  discharged  intermittently,  and  for  this  reason 
repeated  examination  is  necessary.  Chronic  urinary  carriers  have  been 
reported  by  Prigge,90  Houston,91  and  others,  and  are  usually  associated 
with  some  pathological  lesion  of  the  genito-urinary  tract.  In  a  case 
reported  by  Mayer  and  Ahreiner  92  there  was  a  pyonephrosis,  and  in 
other  cases,  cystitis,  or  other  inflammations  of  the  bladder,  ureter,  and 
kidney  have  been  found. 

By  so-called  healthy  carriers  are  meant  individuals  who  harbor 
typhoid  bacilli  in  the  stools  and  in  .whom  no  history  of  typhoid  infection 
at  any  time  in  their  lives  can  be  obtained.  We  know  more  about  typhoid 
fever  than  we  did  formerly,  and  we  think  that  it  is  quite  clear  to  most 
students  of  the  disease  that  a  negative  history  of  this  kind,  especially  if 
obtained  from  people  who  have  lived  vigorous,  active,  physical  lives, 
must  be  very  unreliable.  Extremely  mild  cases  of  typhoid  fever,  while 
not  common,  do  occur,  and  it  is  not  impossible  that  an  individual  with 
an  unusual  resistance  may  have  been  ill  for  a  few  weeks,  perhaps  with  a 
little  fever  at  sometime  without  going  to  a  doctor.  On  the  other 
hand,  Scheller93  in  examining  a  group  of  people,  in  connection  with  the 
investigations  made  of  a  mild  epidemic,  found  a  considerable  number  of 
temporary  carriers  who  did  not  develop  the  disease.  These  people  had 
taken  milk  infected  from  a  carrier,  18  of  a  total  of  44  acquiring  the 
organisms  without  getting  sick,  while  32  of  the  same  group  actually  got 
sick.  It  is  not  impossible,  therefore,  that  individuals  associated  with 
typhoid  cases  and  during  epidemics  may  become  temporary  carriers. 

Carriers  may  increase  enormously  in  the  course  of  epidemics  espe- 
cially if  these  epidemics  take  place  among  the  large  groups  of  vaccinated 
people.  Such  conditions  prevailed  among  the  Allied  and  probably 
among  the  German  Armies  during  the  war,  when  the  enormously  in- 
creased opportunities  for  fecal  transmission  produced  incident  to  active 
warfare,  with  open  latrines,  unprotected  kitchens,  unlimited  fly  breeding, 
and  defective  scattered  small  water  supplies,  made  sanitary  control 
impossible.  Hundreds  of  thousands  of  men  suffered  from  diarrheas  and 

89  Garbat,  Jour.  A.  M.  A.,  Nov.,  1916,  1493. 

90  Prigge,  Klin.  Jahr.,  22,  1909-1910,  245. 

91  Houston,  with  Irwin,  Lancet,  1,  1909,  311. 

92  Mayer  and  Ahreiner,  cited  from  Gay,  loc.  cit. 

93  Scheller,  Cent,  f .  Bakt.,  Erste  Abt.,  Orig.,  45,  1908,  385. 


674  PATHOGENIC   MICROORGANISMS 

mild  intestinal  disease,  without,  or  with  very  slight  febrile  manifesta- 
tions, and  the  investigations  of  many  bacteriologists,  as  well  as  our  own, 
show  that  a  considerable  percentage  of  these  people  were  actually 
infected  with  organisms  of  the  typhoid,  paratyphoid,  and  dysentery 
groups. 

As  to  the  relative  importance  of  the  typhoid  carrier  in  the  morbidity 
of  typhoid  fever,  it  is  very  difficult  to  adduce  accurate  data.  It  is 
pretty  safe  to  say  that  the  carrier  is  growing  relatively  more  important, 
will  in  the  future  probably  be  the  chief  source  of  typhoid  morbidity 
in  well  sanitated  communities,  and  is  the  only  stumbling  block  which 
will  probably  prevent  the  complete  eventual  eradication  of  the  disease. 
Of  recent  years,  as  water,  milk,  and  food  supplies  are  coming  more  and 
more  directly  under  the  vigilant  eyes  of  health  authorities,  the  estimates 
of  the  percentage  of  cases  due  to  carriers,  as  contrasted  with  other 
sources  of  infection,  is  growing  larger  and  larger. 

PATHOLOGICAL  CONSEQUENCES  OF  THE  CARRIER  STATE. — Perhaps 
the  most  common  sequelum  of  the  chronic  carrier  state  is  cholelith- 
iasis. According  to  Exner  and  Heyworski  94  typhoid  bacilli  have  a 
particular  property  of  decomposing  the  bile  salts,  giving  rise  to  a  pre- 
cipitation of  cholesterin,  and  Dorr  95  experimentally  produced  small 
concretions  in  the  gall  bladder  of  infected  animals.  Typhoid  bacilli 
have  often  been  found  in  gall  stones.  Furthermore,  obstruction  of  the 
bile  and  stagnation  due  to  inflammatory  processes  may  be  indirectly 
responsible  for  stone  formation. 

It  is  probable  that  typhoid  carriers  possess  an  especially  high 
resistance  to  second  attacks,  higher  even  than  that  of  the  ordinary 
individual  who  recovers  without  developing  the  carrier  state.  Kuster 
reports  that  of  800  chronic  carriers  observed  in  the  military  hospital  at 
Cologne  during  two  and  one-half  years,  not  a  single  clinical  disturbance 
attributable  to  the  typhoid  bacillus  could  be  determined.  The  occur- 
rence of  cystitis,  pyelitis  and  renal  stones  in  typhoid  carriers  is  not  par- 
ticularly common. 

Occasionally,  typhoid  carriers  may  possess  -agglutinins  and  other 
antibodies  in  the  blood  higher  than  normal.  Lentz  examined  a  number 
of  chronic  carriers  and  found  positive  Widals  in  10  out  of  11;  however, 
only  in  dilutions  of  1  :  20.  Gaethgens  96  found  both  agglutinins  and 
opsonins  higher  in  chronic  carriers  than  in  normal  people,  but  Schone  97 

94  Exner  and  Heyworski,  Wien.  klin.  Woch.,  1908,  7. 

95  Don',  cited  from  Klistcr,  loc.  cit. 

96  Gaethgens,  Deut.  med.  Woch.,  1907,  1337. 

97  Schone,  Munch,  med.  Woch.,  1908,  1063. 


BACILLI    OF    THE    COLON-TYPHOID-DYSENTERY    GROUP     075 

found  no  increase  in  complement  fixation.  In  general,  we  would  not 
hope  for  very  much  light  from  serological  investigations  upon  the  ques- 
tion of  whether  or  not  an  individual  was  a  carrier,  but  would  imme- 
diately proceed  to  bacteriological  examination,  either  by  feces,  urine,  or 
duodenal  examination.  , 

THE  TREATMENT  AND  CURE  OF  TYPHOID  CARRIERS. — The  great 
importance  of  the  typhoid  carrier  from  the  epidemiological  point  of  view 
has  led  to  innumerable  medical  and  surgical  attempts  at  cure.  That 
there  can  be  no  doubt  about  the  possibility  of  cure  in  most  cases,  by 
surgical  gall-bladder  extirpation,  is,  of  course,  certain.  It  will  be  neces- 
sary in  the  future,  however,  especially  on  the  basis  of  the  recent  work  of 
Nichols,  Garbat  and  some  German  observers,  to  precede  such  operations 
by  fecal  and  duodenal  examinations,  and  it  must  be  remembered  that 
there  will  always  be  a  certain  percentage  of  cases  which  are  liver-duct 
carriers,  in  contrast  to  gall-bladder  foci,  which  will  not  clear  up.  Also, 
the  operation  is  not  without  danger,  with  a  certain  amount  of  mor- 
tality, and,  of  course,  cannot  be  applied  generally  upon  the  enormous 
numbers  of  carriers  that  exist. 

According  to  Garbat  and  others,  attempts  to  cure  by  other  means 
necessarily  depend  to  a  veiy  large  extent  upon  early  diagnosis  of  the 
carriers  before  the  condition  has  become  stubbornly  chronic.  But  in  all 
cases,  cure  by  other  than  surgical  means  has  been  discouraging.  Many 
different  methods  have  been  attempted.  Vaccination  with  the  ordi- 
nary typhoid  vaccines  has  given  discouraging  results  in  the  hands  of 
Park  and  many  others,  although  much  was  hoped  from  it  at  first. 
Irwin  and  Houston  98  claimed  to  have  cured  a  urinary  carrier  by  vacci- 
nation, but  Houston  and  Thomas  "  failed  in  other  cases.  Petruski  10° 
in  1902  claimed  that  vaccination  during  the  course  of  the  disease  might 
prevent  the  development  of  the  carrier  state.  But,  on  the  whole, 
vaccination  has  not  brought  the  results  that  have  been  hoped  for  it, 
and  we  are  rather  reluctant  to  believe  that  on  a  theoretical  basis  it  is 
encouraging,  since  the  organisms  in  the  chronic  carrier  are  physiologically 
outside  the  body,  and  not  in  contact  with  antibodies  or  leucocytes. 

Medicinal  treatment  has  been  tried  with  many  agents,  but  without 
much  success.  Discouraging  results  have  been  obtained  with  urotropin, 
methylene  blue,  saliciyates,  iodin  and  arsenic  preparations.  Conradi 101 
in  1910  tried  chloroform  with  apparently  successful  results  in  rabbits 

98  Inrit/  and  Ifonxton,  Lancet,  1,  1909,  154. 

99  HouKlon  and  Tlunmi*,  Cent.  f.  Bakt.  Rcf.,  45,  1910,  390. 

100  Petmski,  cited  rrom  Kiister,  loc.  cit. 

101  Conradi,  Centralb.  f.  Immimit.,  7,  1910,  158. 


676  PATHOGENIC   MICROORGANISMS 

experimentally  converted  into  typhoid  carriers.  Bully 102  tried  this 
treatment  upon  human  carriers,  giving  0.5  c.c.  of  chloroform  in  capsules 
four  times  a  day  for  twenty  days,  without  any  results.  Neosalvarsan 
has  been  tried  without  effect.  The  only  encouraging  reports  we  can 
find  in  an  extensive  review  of  the  literature  to  date  are  the  recent  ones  of 
Kalberlah  103  who  administered  tincture  of  iodine  together  with  animal 
charcoal,  and  those  of  Geronne  104  who  similarly  combined  charcoal 
with  thymol.  None  of  these  methods  have,  however,  been  sufficiently 
confirmed  to  encourage  great  hope. 

THE  TYPHOID  BACILLUS  IN  TRANSIT  FROM  SOURCE  TO  VICTIM. — 
The  typhoid  bacillus  which  reaches  the  outer  world  in  the  feces  and 
urine  of  carriers  and  cases  is  fortunately  not  a  very  resistant  organism. 
It  requires  moisture  and  a  favorable  temperature  approaching  37.5°  C. 
for  multiplication,  and  suitable  nutritive  material.  These  conditions 
being  unfavorable,  it  is -subjected  to  a  rapid  diminution  in  concentration 
by  dilution,  and  dies  out  with  relative  speed.  In  sewage  and  feces, 
moreover,  it  is  subject  to  rapid  destruction  in  the  competition  with  the 
more  hardy  plebeians  with  which  it  comes  in  contact.  In  feces  the 
organisms  will  live  for  very  variable  periods  according  to  temperature 
and  conditions  governing  decomposition.  They  may  be  destroyed 
within  a  day  or  two,  and  in  cesspools,  etc.,  where  they  are  immediately 
mixed  with  large  numbers  of  decomposing  feces,  they  live  for  probably 
not  longer  than  a  few  days  under  any  conditions.  If  feces  are  frozen, 
that  is,  deposited  in  the  open  in  the  winter,  the  organisms  may  live 
throughout  the  winter  and  enter  water  sheds,  etc.,  with  the  thaw.  In 
water,  as  a  rule,  they  do  not  live  more  than  a  few  days  or  perhaps  a 
week,  and  according  to  Rosenau  they  live  longer  in  clean  than  in  con- 
taminated water.  In  sewage  their  life  is  short.  Freezing  does  not  kill 
them.  According  to  the  investigations  of  Gartner105  the  typhoid 
bacilli  could  be  found  in  the  flowing  water  of  the  Paris  water  supply 
after  a  day  and  one-half.  In  all  statements  of  this  kind  it  must  be 
remembered  by  the  sanitarian  that  no  absolute  rules  can  be  set  up, 
since  we  know  that  the  inability  of  all  microorganisms  like  the  typhoid 
bacillus  depend  very  delicately  upon  temperature,  nutrition,  the  presence 
of  other  bacteria,  moisture,  heat,  light  reaction,  etc.  We  have  ourselves 
seen  typhoid  bacilli  alive  and  viable  after  many  years  of  sealing  in  agar 
cultures  preserved  in  the  dark  and  in  a  cool  place,  and,  while  in  nature 

102  Bully,  Zeit.  f.  Hyg.,  61,  1911,  29. 
^Kalberlah,  Med.  Klinik.,  1915. 

104  Geronne,  Berl.  klin.  Woch.,  1915. 

105  Gartner,  Klin.  Jahrb.,  9,  1902. 


BACILLI   OF   THE   COLON-TYPHOID-DYSENTERY    GROUP      677 

the  organisms  disappear  with  relative  speed,  no  rule  can  be  set  up. 
Klein  106  claims  to  have  found  typhoid  bacilli  alive  in  natural  waters 
for  as  long  as  thirty-six  days. 

Since  the  organisms  can  remain  in  the  soil  for  limited  periods  of  time, 
unwashed  vegetables,  salads,  etc.,  are  dangerous,  and,  as  we  shall  see, 
oysters  grown  near  sewage  outlets,  may  also  be  sources  of  infection. 

CHANNELS  OF  TRANSMISSION. — Suspicion  of  typhoid   infection  by 
means  of  water  supplies  dates  back  to  the  early  writing  of  the  English 
physician,  Budd,  in  1856,  who  not  only  believed  that  sewage  contam- 
inated water  conveyed  the  disease,  but  suggested  that  the  origin  of  this 
pollution  lay  in  human  feces.     Since  that  time,  of  course,  bacteriological 
investigation  and  sanitary  water  purification  on  a  large  scale  has  indis- 
putably proven  the  danger  of  water  supplies  in  this  respect.     In  a  few 
cases,  direct  proof  of  typhoid  bacilli  in  the  water  supply  has  been 
brought,  but,  as  a  rule,  indirect  proof  has  had  to  be  adduced,  since 
the  rapid  dilution,  the  usual  lateness  of  water  investigation  after  the 
occurrence  of  cases,  and  the  many  agencies  which  lead  to  the  destruc- 
tion of  typhoid  bacilli  in  water  supplies,  has  made  it  extremely  difficult 
to  find  the  organisms  in  the  water.     Indirect  evidence,  however,  has 
been  sufficiently  convincing  in  that  colon  bacillus  tests  have  revealed 
massive  human  feces  contamination  of  water  to  which  typhoid  infection 
could  be  epidemiologically  traced.     Also,  in  many  localities  the  direct 
diminution  of  typhoid  fever  in  a  community  after  purification  of  the 
water  supply  has  left  little  room  for  doubt.     Thus,  in  Schuder's107  inves- 
tigations of  640  epidemics,  72  per  cent  were  directly  traceable  to  water. 
Water  was  unquestionably  in  former  years  the  most  important  means 
of  the  conveyance  of  typhoid  fever  when  it  occurred  in  definite  epi- 
demics, and  whenever  typhoid  cases  occur  in  any  considerable  number 
in  cities  and  towns,  the  water  supply  must  first  be  excluded  as  a  source 
of  infection.     It  must  also  always  be  taken  into  consideration  when 
typhoid  fever  occurs  in  country  districts  where  small  well  supplies 
are  the  chief  sources  of  drinking  water.     In  Schiider's  statistics,  110  of 
his  640  epidemics  could  be  indirectly  traced  to  milk.     Gay76  states 
that  in  the  rural  communities  typhoid  fever  has  remained  more  or  less 
stationary  during  the  last  ten  years,  while,  in  the  cities,  owing  probably 
to   water-supply   supervision,   it  has  been   diminishing   progressively. 
The  methods  of  examining  water  under  such  circumstances  will  be 
detailed  in  the  section  on  water,  where  emphasis  will  also  be  placed 

lofi  Klein,  Medical  Officers  Report,  Local  Govern.  Board. 
107  Schiider,  Zeit.  f.  Hyg.  u.  Infec.,  38,  1901,  343. 


678  PATHOGENIC   MICROORGANISMS 

upon  tnc  fact  that  bacteriological  water  examinations  of  this  kind  must 
always  be  associated  with  careful  sanitary  survey  of  the  water  shed  and 
engineering  examination  of  the  purification  plant,  if  there  is  one  avail- 
able. It  is  important  for  the  sanitarian  to  remember  that,  while  water 
epidemics  are  constantly  diminishing  as  large  scale  water  purification 
becomes  more  and  more  universal,  there  are  still  occasional  epidemics 
in  which  accidents  have  occurred  to  ordinarily  properly  functioning 
purification  plants. '  Such  an  epidemic  was  recently  reported  from 
Salem,  Ohio,108  where  in  September  and  October  of  1920,  following  a 
rainy  period,  enteritis  of  unknown  origin  afflicted  about  one-half  the 
population.  Subsequent  investigation  showed  that  3  cases  of  typhoid 
fever  had  occurred  in  early  September;  and  typhoid  fever  reports  began 
in  late  October  and  early  November.  Investigation  of  the  water  supply 
revealed  pollution  probably  due  to  the  contamination  of  one  of  the 
gravity  lines  connecting  a  group  of  wells  with  the  reservoir.  Aside 
from  the  earliest  cases  mentioned  above,  the  first  cases  appeared  about 
October  1st,  and  reached  a  peak  of  54  new  cases  on  November  1st, 
which  was  the  highest  daily  number  of  the  epidemic.  Up  to  November 
20th,  a  total  number  of  785  cases,  with  12  deaths  occurred.  Recently, 
we  have  heard  of  another  epidemic  which  occurred  in  a  California  town, 
where  a  small  explosive  outbreak  of  typhoid  fever  occurred  owing  to 
accident  to  the  water  supply  followed  by  direct  pumping  from  the 
river,  for  one  day,  necessitated  by  repairs.  The  considerable  and 
unexceptional  diminution  of  typhoid  fever  in  all  cities  where  water 
supply  purification  plants  have  been  installed,  may  be  found  tabulated 
in  such  books  as  Rosenau's  Hygiene,  Mason's  book  on  water  supply, 
and  others. 

Milk  may  act  as  a  distributor  of  typhoid  fever  either  by  direct  infec- 
tion of  the  milk  from  milk  handlers  who  are  carriers,  or  from  bottles 
that  are  returned  from  houses  where  typhoid  fever  or  typhoid  carriers 
exist.  A  considerable  number  of  milk  epidemics  have  been  traced 
beyond  doubt,  and  have  usually  been  characterized  by  an  explosive 
onset  and  by  the  fact  that  the  majority  of  the  patients  were  women  and 
children.  Milk  is  an  excellent  culture  media  for  the  typhoid  bacillus, 
and  an  enormous  increase  of  the  organisms  in  the  milk  between  contam- 
ination and  consumption  may  occur  without  visible  changes  in  the  milk. 
Uncooked  vegetables,  salad,  radishes,  etc.,  may  be  responsible  for  typhoid 
infection,  and  of  recent  years  it  has  also  been  shown  that  oysters  may 

108  Jour,  of  the  A.  M.  A.,  75,  1920,  1498. 


BACILLI   OF   THE    COLON-TYPHOID-DYSENTERY    GROUP     679 

be  a  source  of  danger.  Conn  109  was  the  first  to  suggest  this,  tracing 
an  epidemic,  which  broke  out,  to  this  cause.  Experiments  by  Foote  no 
showed  that  typhoid  bacilli  may  be  found  alive  in  oysters,  within  three 
weeks  after  they  had  disappeared  from  the  surrounding  water.  While 
there  is  very  little  question  as  to  the  possibility  of  this  form  of  infection, 
it  probably  does  not  occur  very  often.  In  the  investigations  of  Rose- 
nau,  Lurnsden  and  Kastle  m  it  was  found  to  be  a  negligible  factor 
in  the  cases  of  typhoid  fever  occurring  in  the  District  of  Columbia. 

That  flies  play  a  very  important  role  in  the  carrying  of  typhoid 
bacilli  from  feces  to  food,  was  suggested  by  Vaughan  and  by  Veeder  112 
in  1898.  Vaughan  studied  this  particularly,  and  showed  that  in  the 
Army  camps  in  1898,  flies  flew  directly  from  the  latrines  to  the  kitchen; 
in  fact,  he  found  hypochlorite  of  lime  on  the  food,  picked  up  by  the  flies 
in  the  latrines.  During  the  late  war,  there  can  be  very  little  question 
about  the  fact  that  the  enormous  morbidity  of  intestinal  diseases  which 
occurred  in  the  Allied  Armies  at  various  times,  and  especially  during 
the  July  offensive  at  Chateau  Thierry,  was  caused  by  open  latrines  and 
flies,  typhoid  epidemics  being  avoided  only  by  the  universal  vaccination 
of  the  Armies. 

As  water  supplies,  milk  supplies,  etc.,  are  being  supervised  and, 
therefore,  excluded  as  sources  of  typhoid  infection,  contact  infection  is 
becoming  more  and  more  important.  As  a  matter  of  fact,  the  recent 
studies  of  typhoid  morbidity  seem  to  indicate  that  contact  infection  is 
growing  to  be  the  chief  problem  in  the  prevention  of  typhoid  fever. 
Frosch  who  analyzed  978  cases,  concluded  that  65.6  per  cent  were  con- 
tact infections,  and  Drigalski  makes  similar  estimates.  Such  infections 
may  be  from  individual  to  individual  by  close  contact.  They  may  be 
from  cook  and  kitchen  personnel,  to  raw  food  to  consumer.  Instances 
of  such  infection  are  frequent,  the  most  famous  one  being  that  of 
''Typhoid  Mary"  who  was  recently  made  the  subject  of  a  special  pub- 
lication by  Soper.113  This  woman,  a  cook,  worked  for  eight  families  in 
the  course  of  ten  years,  during  which  time  7  outbreaks  directly  trace- 
able to  her  occurred.  Since  that  time  her  movements  from  place  to 
place 'have  usually  been  followed  by  circumscribed  epidemics.  Again, 

109  Conn,  Medical  Record,  December,  1894. 

110  Foote,  Medical  News,  1895. 

111  Rosenau,  Lumsden  and  Kastle,  Pub.  No.  52,  Hyg.  Lab.  U.  S.  Pub.  Health 
Serv.,  190S. 

112  Veeder,  Medical  Record,  45,  1898. 

113  Soper,  Military  Surgeon,  45,  1919,  1. 


680  PATHOGENIC  MICROORGANISMS 

contact  infection  may  take  place  from  fomites — fingers — food  to  mouth, 
that  is,  towels,  bed  clothing,  underclothing,  etc.,  and  emphasizes  the 
importance  of  sanitary  paper  towels,  etc.,  in  toilets. 

Since  in  contact  infection  the  case  is  of  relatively  little  danger, 
largely  because  of  the  fact  that  danger  of  a  case  is  so  well  recognized 
and  precautions  against  transmission  from  such  a  source  are  easily 
taken,  and  have  become  matters  of  routine  in  well-regulated  sick-rooms 
and  hospitals,  the  interest  in  these  infections  centers  upon  the  typhoid 
carrier. 

The  Prevention  of  Typhoid  Fever.114 — The  measures  which  are 
necessary  for  the  prevention  of  typhoid  fever  can  be  easily  deduced 
from  a  consideration  of  the  material  in  the  foregoing  paragraphs. 

Of  great  importance  is  recognition  of  cases,  hospitalization  and  isola- 
tion. During  such  hospitalization  there  should  be  careful  attention  to 
disinfection  of  discharges,  sterilization  of  bedding,  bed-pans,  eating 
utensils,  etc.,  etc.  Patients  should  never  be  discharged  from  hospitals 
until  the  urine  and  feces  have  been  found  free  from  typhoid  bacilli, 
and  several  examinations  at  intervals  of  two  or  three  days  should 
be  negative  before  this  is  considered  to  be  the  case.  .  . 

Attention  to  sewage  disposal,  water  supplies,  filtration  and  chlorina- 
tion  of  water  with  constant  supervision  of  such  plants  from  both  a  bac- 
teriological, chemical  and  engineering  point  of  view. 

Similar  supervision  of  milk  supply,  with  especial  attention  to  the 
carrier  state  of  the  personnel  of  dairies  and  milk  handlers. 

Public  health  arrangements  for  the  immediate  epidemiological  study 
of  cases  which  occur  and  laboratory  facilities  for  the  tracing  of  carriers 
indicated  by  such  epidemiological  studies. 

Eventual  examination  for  the  carrier  state  of  food  handlers,  pro- 
fessional cooks,  and  exclusion  from  such  professions  of  people  found  to  be 
carriers. 

Community  measures  for  the  suppression  of  fly-breeding  places  and 
flies,  screening  of  kitchens,  and  the  absolute  elimination  of  open  latrines 
of  any  kind. 

The  prevention  of  oyster  culture  near  sewage  outlets. 

Finally,  more  and  more  attention  must  be  given  to  generalized 
vaccination. 

Vaccination  and  Specific  Therapy  in  Typhoid  Fever. — The  failure 
to  produce  a  soluble  toxin  from  typhoid  cultures  has  naturally  so  far 

114  See  Rosenau's  Hygiene — Also  Zinsser — Prevention  of  Com.  Dis.  in  Nelson's 
System,  1921. 


BACILLI    OF    THE    COLON-TYPHOID-DYSENTERY    GROUP     681 

precluded  the  possibility  of  an  antitoxin  therapy,  such  as  that  which  has 
been  successful  in  diphtheria.  In  the  light  of  our  present  knowledge 
of  the  poisonous  products  of  the  typhoid  bacillus  it  seems  but  natural 
that  attempts  by  earlier  investigators  to  apply  the  -principles  of  Beh- 
ring's  work  to  typhoid  fever  were  doomed  to  fail.  Attempts  to  employ 
specific  bactericidal  and  bacteriolytic  sera  for  therapeutic  purposes  in 
this  disease  have  also  been  without  favorable  result. 

Active  Prophylactic  Immunization. — We  have  seen  that  work  by 
Pfeiffer  and  Kolle  and  later  by  many  others  has  shown  that  it  is  com- 
paratively easy  to  immunize  animals  actively  against  typhoid  infection 
by  the  sytematic  injection  of  graded  doses,  at  first  of  dead  bacilli,  later 
of  fully  virulent  live  cultures.  Attempts  to  apply  these  principles  pro- 
phylactically  have  been  made  recently  on  a  large  scale  by  Wright  and 
his  associates  upon  English  soldiers  in  South  Africa,  and  by  German 
observers  in  East  Africa. 

The  first  recorded  experiment  of  this  sort  which  was  done  upon 
human  beings  was  that  of  Pfeiffer  and  Kolle,115  who  in  1896  treated  two 
individuals  with  subcutaneous  injections  of  an  agar  culture  of  typhoid 
bacilli  which  had  been  sterilized  at  56°  C.  The  first  injection  was  made 
with  2  cmm.  of  this  culture.  Three  or  four  hours  after  the  injection  the 
patient  suffered  from  a  chill,  his  temperature  gradually  rose  to  105°  F., 
and  there  was  great  prostration  and  headache,  but  within  twenty-four 
hours  the  temperature  had  returned  to  normal. 

This  experiment  showed  that  such  injections  could  be  practiced  upon 
human  beings  without  great  danger. 

Simultaneously  with  the  work  of  Pfeiffer  and  Kolle,  Wright116 
conducted  similar  experiments  on  officers  and  privates  in  the  English 
army. 

The  actual  number  of  persons  treated  directly  or  indirectly  under 
Wright's  117  supervision  in  an  investigation  covering  a  period  of  over  four 
years  comprised  almost  one  hundred  thousand  cases.  The  methods 
employed  by  Wright  have  been  modified  several  times  in  minor  details; 
the  principles,  however,  have  remained  consistently  the  same.  In  the 
first  experiments  Wright  employed  an  agar  culture  three  weeks  old, 
grown  at  37°  C.,  then  sterilized  at  a  temperature  below  60°  C.,  and  pro- 
tected from  contamination  by  the  addition  of  five-tenths  per  cent  of 

115  Pfeiffer  und  Kolle,  Deut.  med.  Woch.,  xxii,  1896,  xxiv,  1898. 

116  Wright,  Lancet,  Sept.,  1896. 

117  Wright  and  Semple,  Brit.  Med.  Jour.,  1897;  Wright  and  Leishmann,  Brit.  Med. 
Jour.,  Jan.,  1900. 


682  PATHOGENIC   MICROORGANISMS 

carbolic  acid.  Later,  Wright ll8  employed  bacilli  grown  in  a  neutral 
1-per-cent  pepton  bouillon  in  shallow  layers  of  flasks. 

Great  importance  is  attached  both  to  the  virulence  of  the  typhoid 
strain,  which  may  to  a  moderate  extent  be  standardized  by  passage 
through  guinea-pigs,  and  to  care  in  using  low  temperatures  for  final 
sterilization.  The  temperature  recommended  by  Harrison,  is  52°  C. 
after  which  the  cultures  are  carbolized. 

It  is  nevertheless  extremely  difficult  to  tabulate  satisfactory  statis- 
tics from  a  mass  of  experiments  observed  by  a  large  number  of  indi- 
viduals. On  the  whole,  however,  it  seems  fair  to  state  that  advan- 
tageous results  followed  the  active  immunization  practiced  by  Wright. 
Wright's  own  estimation,  in  a  careful  attempt  to  present  the  subject 
fairly,  gives  a  reduction  of  the  morbidity  from  typhoid  fever  in  the 
British  army  of  50  per  cent,  and  a  reduction  of  the  mortality  of  those 
who  became  infected  in  spite  of  inoculations  of  50  per  cent  also.  It  is 
not  at  all  impossible  that  a  number  of  different  strains  will  have  to  be 
used  eventually  for  the  ideal  vaccine,  inasmuch  as  the  antigenic  differ- 
ences which  have  been  recently  discovered  would  make  it  seem  that  no 
single  strain  can  be  expected  to  produce  antibodies  which  would  protect 
against  all  other  strains.  It  is  not  impossible  that  some  individual 
strain  may  combine  the  antigenic  properties  of  the  entire  group.  This, 
however,  has  still  to  be  worked  out. 

The  method  of  Pfeiffer  and  Kolle  consists  in  the  injection  of  salt- 
solution  emulsions  of  fresh  agar  cultures  sterilized  at  60°  C.  The 
results  reported  were  in  general  favorable. 

Extensive  tests  in  the  United  States  Army,  observed  by  Russell,119 
have  removed  any  doubt  which  may  have  existed  as  to  the  efficacy  of 
prophylactic  typhoid  vaccination.  Russell's  statistics  show  a  steady 
decline  of  typhoid  in  the  U.  S.  Army  beginning  with  the  introduction 
of  compulsory  vaccination  in  1910.  In  1913  there  was  but  one  case 
among  over  80,000  men. 

The  method  at  present  employed  is  as  follows:  The  "Rawlings" 
strain  of  typhoid,  obtained  from  Wright,  is  used.  Eighteen-hour  agar 
cultures  in  Kolle  flasks  are  washed  off  with  sterile  saline  to  an  approxi- 
mate concentration  of  one  billion  to  the  cubic  centimeter.  The  sus- 
pension is  killed  at  53°  C.  for  one  hour  and  0.25  per  cent  tricresol  is 
added.  Aerobic  and  anaerobic  culture  controls  are  made  and  a  rabbit 

118  Wright,  Brit.  Med.  Jour.,  1901;   Lancet,  Sept.,  1902;   Brit.  Med.  Jour.,  Oct., 
1903. 

119  Russell,  Am.  Jour,  of  Med.  Sc.,  cxlvi,  1913. 


BACILLI   OF   THE    COLON-TYPHOID-DYSENTERY    GROUP     683 

and  mouse  inoculated  to  insure  sterility.  For  immunization  3  to  4 
doses  are  given  ranging  in  quantity  from  500  million  to  one  billion  at 
seven-  to  ten-day  intervals.  The  protection  probably  lasts  about  two 
years,  though  this  is  not  certain. 

Another  point  of  importance  in  this  connection  has  recently  been 
raised  by  Metchnikoff  and  Besredka.120  They  vaccinated  chimpanzees 
with  typhoid  bacilli  and  found  that  when  emulsions  of  the  clear  bac- 
teria were  used,  protection  was  only  slight.  Better  results  were  obtained 
—that  is,  apparently  complete  protection  within  eight  to  ten  days — 
when  living  sensitized  bacteria  were  injected  (bacteria  which  had  been 
exposed  to  the  action  of  inactivated  immune  serum.)  Broughton  has 
applied  this  method  to  human  beings.  Gay76  has  also  prepared  a  sen- 
sitized dead  typhoid  vaccine  which  he  has  used  in  a  considerable 
number  of  cases.  It  will  take  some  time,  however,  before  a  statistical 
estimation  of  the  superiority  of  this  method  over  the  older  vaccination 
with  dead  bacteria  will  be  possible. 

Gay76  sensitizes  his  bacilli  with  strong  immune  serum,  precipitates 
with  alcohol,  dries  and  grinds  them  into  powder,  and  uses  weighed 
amounts  of  the  powder. 

During  recent  years  in  France  another  form  of  vaccine  has  been 
used  which  has  advantages  which  lead  to  its  general  adoption,  if  its  im- 
munizing value  can  be  successfully  proven.  This  is  the  so-called  "lipo- 
vaccine."  It  consists  of  typhoid  cultures  grown  on  agar,  taken  up  in  salt 
solution,  the  sediment  partially  dried,  and  then  shaken  up  with  olive  oil, 
the  formula  for  which  is  not  available.  It  can  also  be  produced  by  grind- 
ing up  the  typhoid  sediment  with  lanolin  and  shaking  up  this  well-ground 
paste  with  sterile  olive  oil.  Great  difficulty  has  been  encountered  in  the 
sterilization  of  this  vaccine,  a  matter  which  still  needs  much  investiga- 
tion. The  vaccine  has  the  advantage  of  giving  much  diminished  reac- 
tions, and  it  is  claimed  that  three  times  the  amounts  given  in  the 
ordinary  saline  suspensions  can  be  given  without  serious  discomfort  to 
the  patient.  There  is  probably  slow  absorption  of  this  vaccine  and  it  is 
claimed  that  a  single  dose,  because  of  the  slow  absorption  of  the  organ- 
isms, may  be  sufficient  to  vaccinate. 

During  the  recent  war,  typhoid  vaccination  has  thoroughly  justified 
itself.  We  are  not  in  possession  at  the  present  writing,  of  consolidated 
reports  of  all  the  European  Armies,  but  the  Surgeon  General's  report  for 
the  United  States  Army,  published  in  1919,  shows  the  magnificent  results 
obtained  by  vaccination  in  American  troops.  During  the  pre-vaccination 

120  Metchnikoff  and  Besredka,  Am.  dc  Tinst.  Past.,  1911. 


684  PATHOGENIC   MICROORGANISMS 

days  in  the  Civil  War  and  the  Spanish  American  War,  the  admission 
rates  for  typhoid  fever  were  enormous.  During  the  first  year  of  the 
Civil  War  the  annual  admssion  rate  was  70.69,  with  a  death  rate  of  19.61, 
and  it  is  likely  that,  in  addition  to  this,  a  large  number  of  unrecognized 
cases  occurred.  During  the  Spanish  American  War  and  the  Philippine 
Insurrection  in  the  years  1898  to  1899,  the  annual  admission  was  91.22, 
and  the  death  rate  9.67.  During  the  last  World  War  the  method  of 
vaccination  used  consisted  in  three  inoculations  at  seven-day  intervals, 
of  the  salt  solution  suspension  triple  vaccine,  containing  typhoid 
"Rawlings,"  paratyphoid  A  and  B,  the  first  dose  containing  one-half 
million  bacilli,  and  the  second  and  third  containing  a  billion  each.  The 
typhoid  rate  was  so  low  in  the  camps  in  the  United  States  that  a  young 
man  in  the  camp  had  45  times  less  opportunity  of  getting  typhoid  fever 
than  did  the  same  age  group  in  civilian  life  during  the  same  period. 
Although  approximately  three  million  men  passed  through  the  camps 
during  the  course  of  1918,  the  actual  admission  rate  for  the  United 
States  was  0.17.  In  Europe,  in  spite  of  the  most  terrific  sanitary  condi- 
tions in  some  of  the  battlefields  during  the  summer,  and  with  perhaps 
two  million  troops  in  France,  there  were  only  488  cases  with  88  deaths, 
and  this,  in  spite  of  the  fact,  as  we,  ourselves,  observed,  that  the  oppor- 
tunities for  transmission  were  enormous  in  battle  areas  in  which  sani- 
tation was  practically  impossible,  and  water  supplies  were  bad  and 
could  not  be  corrected. 

The  question  still  remains  as  to  how  long  typhoid  vaccination  can  be 
regarded  as  efficient.  There  is  no  absolute  information  upon  which 
opinions  can  be  based.  Vaccination  is  not  a  complete  protection  at  any 
time,  and  a  recently  vaccinated  individual  may  still  occasionally  con- 
tract the  disease  if  he  is  injected  with  a  large  dose  of  virulent  organisms. 
The  protection,  however,  is  very  powerful  and  will  prevent  contraction 
of  the  disease  from  the  ordinary  chance  infection.  We  should  state  on 
general  information  that  repetition  every  two  years  ought  to  be  suf- 
ficient for  civilian  purposes.  For  the  armies  in  the  field,  we  ourselves 
would  favor  a  first  vaccination  with  three  doses  as  stated  above,  and 
single  or  double  doses  repeated  every  six  months. 

Specific  Treatment  of  Typhoid  Fever.7® — Anti-sera  against  typhoid 
fever  have  been  produced  by  a  large  number  of  workers,  notably  Chante- 
messe 121  and  Besredka  122  both  of  whom  used  the  serum  of  horses 
immunized  with  typhoid  bacilli  or  "endo-toxin,"  so-called.  Garbat 

121  Chantemesse,  Prog,  med.,  7,  1989,  245. 

122  Besredka,  Ann.  Inst.  Past.,  16,  1902,  918. 


BACILLI   OF   THE   COLON -TYPHOID-DYSENTERY   GROUP     685 

and  Meyer  123  believed  that  an  improvement  of  results  could  be  obtained 
by  mixing  the  sera  of  animals  that  had  been  immunized  with  sensitized 
bacteria  and  those  treated  with  normal  typhoid  bacilli. 

At  the  present  time,  however,  practice  has  not  sustained  the  hopes  of 
a  specific  passive  immunization  in  the  treatment  of  typhoid  fever. 

Since  1893,  various  workers  have  tried  to  treat  typhoid  fever  by 
injecting  killed  cultures  or  vaccines  of  typhoid  bacilli.  No  results  of 
importance  were  obtained  until  Ichikawa  124  in  1914  began  to  inject 
dead  typhoid  bacilli,  intravenously.  Gay  and  Claypole  125  and  others 
have  since  taken  up  this  method.  The  intravenous  injection  of  vac- 
cines in  this  way  has  given  most  astonishing  results  in  that  the  injection 
has  usually  resulted  in  a  violent  reaction,  with  often  a  chill  and  sudden 
drop  of  temperature,  and,  subsequently,  very  often  definite  improve- 
ment of  the  cases.  Although  this  method  was  at  first  regarded  as  spe- 
cific by  the  writers  mentioned  above,  Kraus  126  in  1915  showed  that  he 
could  produce  similar  reactions  in  typhoid  patients  with  colon  bacilli, 
as  well  as  with  typhoid  bacilli,  and  similar  observations  were  made  by 
Liidke  127  and  others.  It  is  now  quite  clear  that,  whatever  results  are 
obtained  by  such  treatment  of  typhoid  patients,  they  cannot  be  regarded 
as  specific  reactions  in  the  ordinary  sense  of  the  word. 

123  Garbat  and  Meyer,  Zeit.  f.  exper.  Path.,  8,  1910,  1. 

124  Ichikawa,  Mitteil.  d.  medic.  Gessellsch.  zu.  Tokio,  1914,  28,  H.  21. 

125  Gay  and  Claypole,  Arch.  Inter.  Med.,  12,  1913,  613. 

126  Kraus,  Wien.  klin.  Woch.,  1915,  29. 

127  Liidke,  Munch,  med.  Woch.,  1915,  321. 

*  For  a  thorough  and  clear  treatment  of  the  problem  connected  with  typhoid 
fever,  see  Gay,  Typhoid  Fever,  MacMillan  and  Company,  New  York,  1918. 


CHAPTER  XXXIII 

BACILLI    OF    THE    COLON-TYPHOID-DYSENTERY    GROUP 

(Continued) 

BACILLI   OF   THE   PARATYPHOID— ENTERITIDIS    GROUP 
AND  THE  PARATYPHOID   INFECTIONS 

(Bacilli  of  Meat  Poisoning  and  Paratyphoid  Fever) 

THERE  is  an  extensive  group  of  Gram-negative  bacilli  which  because 
of  their  morphology,  cultural  behavior,  and  pathogenic  properties, 
are  classified  as  intermediate  between  the  colon  and  the  typhoid  types. 
The  microorganisms  belonging  to  this  group  have  been  described,  most 
of  them,  within  the  last  fifteen  years,  but  few  of  them  have  been  fully 
identified  with  one  another.  They  have  been  variously  designated  as  the 
" hog-cholera  group,"  "the  enteritidis  group,"  the  "paracolon  group" 
or  "paratyphoid  group,"  because  of  the  pathological  conditions  with 
which  the  chief  members  under  investigation  have  been  found  associated. 

Attempts  to  systematize  the  group  by  the  comparative  study  of  a 
large  number  of  its  members  have  been  made,  notably  by  Buxton  l 
and  by  Durham,2  and  the  work  of  these  writers,  based  on  cultural  and 
agglutinative  studies,  has  added  materially  to  our  knowledge  of  these 
organisms. 

The  microorganisms  of  this  group  are  morphologically  indistinguish- 
able from  the  colon  and  typhoid  bacilli.  They  are  Gram-negative  and 
possess  flagella.  Their  motility  is  variable,  but  usually  approaches 
that  of  the  typhoid  bacilli  in  activity.  They  correspond,  furthermore, 
to  the  two  other  groups  in  their  cultural  characteristics  upon  broth, 
agar,  and  gelatin.  On  potato  they  vary,  some  of  them  approaching  in 
delicacy  the  typhoid  growth  upon  this  medium,  others  more  closely 
approximating  the  heavy  brownish  growth  of  B.  coli.  Indol  is  rarely 
formed  by  them,  though  this  has  not  been  absolutely  constant  in  all 
descriptions.  As  a  group,  they  are  easily  distinguished  from  Bacillus 

1  Buxton,  Jour.  Med.  Res.  N.  S.,  iii,  1900. 

2  Durham,  Jour.  Exper.  Med.,  v,  1901. 

686 


BACILLI    OF    THE    COLON-TYPHOID-DYSENTERY    GROUP     087 

typhosus  on  the  one  hand,  and  from  Bacillus  coli  on  the  o.ther>  by  the 
following  simple  reactions  tabulated  by  Buxton.3 


B.  Coli. 


Paratyph.  etc. 


B.  Typhosus. 


Coagulation  of  milk -f- 

Production  of  indol 

Fermentation  of  lactose  with  gas -f 

Fermentation  of  dextrose  Wtth  gas 

Agglutination  in  typhoid-immune  serum 


-  (ac.) 


The  simplest  differentiation  between  the  large  groups  can  be  made  by 
fermentation  tests  as  follows : 


. 

Gas  upon 
Dextrose. 

Gas  upon 
Lactose. 

Gas  upon 
Saccharose. 

Gas  on 
Dulcit. 

B  typhosus 

Intermediates  
B.  coli  communis  
B.  coli  communior  

+ 
+ 
+ 

+ 
+ 

+ 

+ 
+ 

B.  acidi  lactici  
B.  lactis  aerogenes  4  

+ 
-}- 

+ 
+ 

+ 

4  Jackson. 

Pathogenically,  the  bacilli  of  this  "  intermediate  group"  have  attracted 
attention  chiefly  in  connection  with  meat  poisoning,  and  with  protracted 
fevers  indistinguishable  from  mild  typhoidal  infections. 

General  Survey  of  the  Group.  —  In  1888,  Gartner5  described  a  bacillus 
which  he  isolated  from  the  meat  of  a  cow,  the  ingestion  of  which  had  produced 
the  symptoms  of  acute  gastrointestinal  catarrh  in  57  people.  One  of  these  died  of 
the  disease  and  the  bacilli  could  be  demonstrated  in  the  spleen  and  blood  of  the 
patient. 

This  bacillus,  called  Bacillus  enteritidis  by  Gartner,  was  actively  motile, 
formed  no  indol,  but  produced  gas  in  dextrose  media.  Acute  gastrointestinal 
symptoms  could  be  induced  by  feeding  the  organisms  to  mice,  guinea-pigs, 
rabbits,  and  sheep,  and  the  bacilli  could  be  recovered  from  the  infected  animals. 
The  bacterial  bodies  themselves  were  found  by  Gartner  to  be  toxic,  containing  a 
poison  which  was  extremely  resistant  to  heat.  Sterilized  cultures  showed  the 
same  pathogenic  effects  as  the  living  bacilli.  Epidemics  of  meat  poisoning  sim- 


3  Buxton,  loc.  cit. 

5  Gartner,  Corresp.  Bl.  d.  Aerzt.  Vereins,  Turingen,  1888. 


688  PATHOGENIC   MICROORGANISMS 

ilar  to  the  one  described  by  Gartner,  in  which  similar  bacteria  were  isolated, 
were  those  described  by  Van  Ermengem,6  occurring  at  Morseele  in  1891,  the  one 
described  by  Hoist,7  the  Rotterdam  epidemic  described  by  Poels  and  Dhont,8 
the  one  described  by  Basenau,  and  many  others. 

Bacillus  Morseele  of  Van  Ermengem,  Bacillus  bovis  morbificans  of  Basenau,9 
and  bacilli  isolated  in  similar  epidemics  by  others,  are,  except  for  slight  differ- 
ences in  minor  characteristics,  almost  identical  with  Gartner's  microorganism. 

In  1893,  Theobald  Smith  and  Moore  10  noted  a  great  similarity  between  the 
so-called  hog-cholera  bacillus,  the  bacilli  of  the  Gartner  group,  and  Bacillus 
lyphi  murium  isolated  by  Loeffler.  These  observers  first  used  the  term  "  hog- 
cholera"  group  for  the  organisms  under  discussion. 

In  1899  Reed  and  Carroll  n  noted  that  Bacillus  icteroides,  associated  by 
Sanarelli  with  yellow  fever,  was  culturally  similar  to  the  bacillus  of  hog 
cholera. 

Meanwhile,  other  observers  had  been  isolating  bacilli,  similar  to  those 
spoken  of  above,  from  cases  of  protracted  fevers  in  human  beings,  often  closely 
simulating  typhoid  infections.  The  first  cases  of  this  kind  on  record  were  those 
of  Achard  and  Bensaude.12 

In  1897,  Widal  and  Nobecourt 13  described  a  bacillus  which  they  had  isolated 
from  an  esophageal  abscess  following  typhoid  fever,  which  closely  resembled 
Bacillus  psittacosis  of  Nocard,14  and  which,  following  a  nomenclature  previously 
suggested  by  Gilbert,15  they  designated  the  paracolon  bacillus.  This  micro- 
organism, isolated  from  a  parrot  by  Nocard,  showed  a  close  resemblance  to 
bacilli  of  the  Gartner  group. 

There  are  a  large  number  of  apparently  nonpathogenic  organisms  sometimes 
referred  to  as  paratyphoid  C,  but  better  perhaps  as  "heterogeneous  types," 
which  are  culturally  identical  with  the  paratyphoid  but  do  not  agglutinate  in 
either  paratyphoid  A  or  B  sera.  An  anti-serum  produced  with  these  types 
usually  reacts  with  the  homologous  strain  only.  Such  organisms  have  been 
studied  by  Krumwiede  and  many  others,  including  ourselves.  Strains  recently 
isolated  at  this  laboratory  came  from  casss  of  nephritis,  German  measles, 
jaundice,  and  the  stools  of  healthy  soldiers,  plated  as  a  matter  of  routine. 

In  1898,  Gwyn16  reported  a  case  at  the  Johns  Hopkins  Hospital,  which  pre- 

6  Van  Ermengem,  Bull,  Acad.  d.  mcd.  de  Belgique,   1892;    "Trav.  de  lab.  de 
Puniv.  de  Gand,"  1892. 

7  Hoist,  Ref.  Cent.  f.  Bakt.,  xvii.  1805. 

8  Poels  und  Dhont,  Holland  Zeit.  f.  Tierheilkunde,  xxiii,  1894. 

9  Basenau,  Arch.  f.  Hyg.,  xx,  1894. 

10  Th.  Smith  and  Moore,  U.  S.  Bureau  of  Animal  Industry  Bull,  vi,  1894. 

11  Reed  and   Carroll,   Medical   News,   Ixxiv,    1899. 

12  Achard  and  Bensaude,  Bull,  de  la  soc.  d.  hopitaux  de  Paris,  Nov.,  1906. 

13  Widal  et  Nobecourt,  Semaine  mod.,  Aug.,  1897. 

14  Nocard,  Ref.  Baumgartcn's  Jahresb.,  1896. 

15  Gilbert,  Semaine  mcd.,  1895. 

16  Gwyn,  Johns  Hopkins  Hosp.  Bull.,  1898. 


BACILLI   OF   THE   COLON-TYPHOID-DYSENTERY   GROUP     689 

sented  all  the  symptoms  of  typhoid  fever,  but  lacked  serum  agglutinating  power 
for  Bacillus  typhosus.  From  the  blood  of  the  patient,  Gwyn  isolated  an  organ- 
ism, with  cultural  characteristics  similar  to  those  of  the  Gartner  bacillus,  which 
he  called  a  "paracolon  bacillus."  This  bacillus  was  agglutinated  specifically 
by  the  serum  of  the  patient. 

Gushing,17  in  1900,  isolated  a  similar  microorganism  from  a  costochondral 
abscess,  appearing  during  convalescence  from  supposed  typhoid  fever. 

In  the  same  year,  Schottmuller  18  reported  five  cases  from  which  similar 
bacilli  were  isolated.  Careful  cultural  studies  of  the  microorganisms  here 
obtained  showed  that  they  could  be  divided  into  two  similar,  yet  distinctly 
different  types,  one  of  them,  the  "Miiller"  organism  (later  "A"  type),  approach- 
ing closely  to  the  typhoid  type,  especially  in  its  growth  upon  potato;  the  other, 
the  "Seeman"  type  (later  "B"  type)  corresponding  more  closely  to  the  Gart- 
ner bacilli.  Similar  cases  were  reported  by  Kurth,19  Buxton  and  Coleman,20 
Libman,21  and  others. 

The  two  types  of  organisms,  paratyphoid  A  and  B  described  by  Schott- 
muller and  studied  by  many  other  observers,  can  be  culturally  differentiated, 
though  not  without  difficulty. 

Differential  Considerations. — The  differentiation  of  the  various 
organisms  within  the  paratyphoid,  enteritidis  group  is  a  very  difficult 
matter.  The  paratyphoid  "A"  organisms  split  off  rather  easily  from 
the  rest  of  them,  and  seem  to  represent  a  fairly  homologous  group.  The 
paratyphoid  "  A"  organisms,  as  shown  by  Ford,22  and  more  particularly 
by  Krumwiede  and  Kohn,23  differ  from  the  rest  of  the  organisms  of  this 
general  group  in  not  fermenting  xylose.  Also,  they  do  not  change  lead 
acetate  agar.  Serologically,  it  may  be  said  in  a  general  way,  though  it 
is  not  absolutely  true,  that  paratyphoid  "A"  represents  an  antigenically 
homologous  group  and  that  a  serum  produced  with  one  member  of  the 
"A"  group  will,  in  a  general  way,  possess  antibodies  against  other  "A" 
organisms.  The  remaining  organisms  of  this  paratyphoid  group  differ 
materially  from  each  other  and  cannot  be  subdivided  into  final  groups, 
as  yet.  However,  a  tentative  grouping,  based  partly  upon  the  sources 
from  which  they  were  obtained  and  partly  upon  fermentations  and 
serological  reactions,  can  be  attempted,  and  a  great  deal  of  valuable 
work  in  this  direction  has  been  done  by  Theobald  Smith  and  his  co- 

17  Gushing,  Johns  Hopkins  Hosp.  Bull.,  1900. 

18  Schottmuller,  Deut.  med.  Woch.,  1900;   Zeit.  f.  Hyg.,  xxvi. 

19  Kurth,  Deut.  med.  Woch.,  1901. 

20  Buxton,  and  Coleman.  Proc.  N.  Y.  Pathol.  Soc.,  Feb.,  1902. 

21  Libman,  Jour.  Med.  Res.,  N.  S.,  iii,  1902. 

22  Ford,  Med.  News,  June  17,  1905. 

23  Krumwiede  and  Kohn,  Jour.  Med.  Res.,  36,  1917,  509. 


690 


PATHOGENIC   MICROORGANISMS 


workers,  by  Smith  and  Ten  Broeck,24,  by  Krumwiedc,  Pratt  and  Kohn,25 
and  many  others.  The  following  fermentation  chart  indicates  briefly 
a  summary  of  the  reactions  of  the  more  common  members  of  this  group, 
chiefly  constructed  according  to  the  work  of  Krumwiede  and  his  co- 
workers. 


Lead 

Dex- 
trose. 

Man- 
nit. 

Lac- 
tose. 

Xylose 

Rham- 
nose. 

Sac- 
cha- 

Dulcit 

Ace- 
tate 

Indol. 

Motil- 
ity. 

rose. 

Agar. 

B.  parat.  "A".  .  . 

© 

© 

_ 

_ 

+ 

_ 

slow 

_ 

C  *»  £3-2 

+ 

B.  parat.  "B".  .  . 

© 

© 

— 

+ 

+ 

— 

+ 

+ 

^^   y  -*-» 

+ 

B.  enteritidis  

© 

© 

— 

-f- 

4. 

— 

-j- 

4. 

c"wi"c  c 

4- 

B.  abortus  equi  .  . 

© 

© 

- 

+ 

+ 

- 

+ 

- 

s  °  v  ° 

+ 

B.  hog  cholera.  .  . 

© 

© 

— 

-f 

-f- 

— 

irreg. 

— 

g^  ^"c+i 

4. 

B.  typhi  murium  . 

© 

© 

- 

+ 

+ 

- 

+ 

S-SjS-S'g 

-j- 

^^  v  '<n  ft 

®  =  acid  and  gas 


—  =  negative      +  =  acid,  no  gas. 


This  table  brings  out  a  very  important  point  namely,  that  xylose 
fermentation  is  one  of  the  sharply  differential  characteristics  between 
paratyphoid  "A"  and  all  the  rest  of  the  group.  Whether  or  not  it  is 
possible  to  differentiate  by  cultural  study  between  the  "B"  organisms 
of  man  and  those  derived  from  different  animal  sources  is  questionable. 
We  do  not  believe  that  it  can  be  done  reliably  at  the  present  time, 
though  certain  studies,  by  the  writers  named,  on  the  quantitative  rela- 
tionship between  reduction  of  fuchsin  and  fermentation  of  dulcite  and 
arabinose  would  hold  out  some  hope  for  future  clarification  of  this 
difficult  problem.  These  writers,  however,  also  call  attention  to  a  fact 
noted  in  a  less  extensive  way  by  us  and  by  many  other  writers,  namely, 
that  agglutination  and  cultural  reactions  do  not  correlate  in  many 
instances  within  this  group.  This  is  a  thing  which  Theobald  Smith 
pointed  out  not  only  for  this  group  but  for  other  groups  of  bacteria 
some  time  ago,  and  would  lead  us  to  be  slow  to  assume  that  either 
agglutination  or  even  cultural  similarity  are  always  to  be  regarded  as 
definitely  signifying  biological  relationship. 

This  opinion,  too,  is  confirmed  by  the  extensive  and  careful  investi- 
gations of  Jordan  which  the  reader  interested  in  this  subject  may  find  in 
the  series  of  papers  published  in  the  Journal  of  Infectious  Diseases, 
Volumes  20,  21,  22,  23  and  26.  Jordan26  finds,  for  instance,  that  the 

24  Smith  and  Ten  Broeck,  Jour.  Med.  Res.,  31,  1915,  503  and  547. 

25  Krumwiede,  Parti  and  Kohn,  Jour.  Med.  Res.,  34,  1916,  34  and  355,  pp.  55,  357 
and  443. 

26  Jordan,  Jour.  Infec.  Dis.,  Vols.,  20,  21,  22,  23  and  26. 


BACILLI   OF   THE    COLON-TYPHOID-DYSENTERY    GROUP     691 

B.  enter! ti  dis  group,  in  other  words,  the  meat  poisoning  group  of 
man,  is  culturally  indistinguishable  from  other  paratyphoid  "B" 
organisms,  but  represents  a  distinctive  agglutinative  group,  at  least  in 
the  strains  obtained  by  him. 

Jordan  adds  in  his  last  paper  of  1920  that  there  is  no  escape  from 
the  conclusion  that  variations  are  common  in  the  paratyphoid-enter- 
itidis  group,  both  as  to  fermentation  and  agglutination.  He  sum- 
marizes that  the  most  permanent  characteristics  of  the  group  are  the 
fact  that  rhamnose  is  fermented  by  all  the  paratyphoids,  but  is  not 
fermented  by  typhoid;  that  paratyphoid  "A"  neglects  to  ferment 
xylose,  whereas  all  the  other  members  of  the  group  do  ferment  xylose. 
Added  to  this  is  the  fundamental  ability  of  the  group  as  a  whole,  includ- 
ing the  typhoid  bacilli,  to  attack  glucose.  These  fermentation  charac- 
teristics are  fundamental,  are  the  least  subject  to  change,  and,  according 
to  Jordan,  are  more  basically  characteristic  than  are  the  agglutinative 
reactions,  • 

PATHOGENICITY  OF  THE  PARATYPHOID  GROUP  FOR 

ANIMALS 

We  have  already  seen  that  organisms  of  this  group  cause  a  con- 
siderable number  of  different  animal  disease. 

B.  HOG  CHOLERA. — It  was  formerly  believed  that  hog  cholera  was 
due  to  the  bacillus  which  bears  this  name,  and  first  described  in  this  con- 
nection by  Salmon  and  Smith,27  but  since  then  it  has  been  found  that 
this  disease  is  due  to  a  filtrable  virus.  The  constant  presence  of  the 
organism  in  animals  suffering  from  this  disease,  is,  therefore,  something 
of  a  mystery,  but  is  probably  due,  as  Dorset  has  suggested,  to  the  fact 
that  the  organism  is  a  constant  inhabitant  of  the  intestinal  canal  in 
hogs,  and  manages  to  get  into  the  circulation  as  a  consequence  of  the 
pathological  conditions  incident  to  hog  cholera.  It  is  worth  noting 
that  a  similar  association  of  organisms  in  blood  cultures,  etc.,  with  dis- 
eases of  which  thejr  are  obviously  not  the  primary  etiological  factor 
has  been  observed  in  other  conditions,  notably,  for  example,  the  Plotz 
bacillus  in  typhus  fever. 

B.  TYPHI  MURIUM. — In  rodents  diseases  caused  by  the  members  of 
the  paratyphoid  group  are  common.  We  have  already  spoken  of 
Loeffler's  discovery  of  the  B.  Typhi  Murium  as  the  cause  of  an  epidemic 
disease  of  mice,  a  condition  which  is  of  the  greatest  annoyance  to 
breeders  of  mice  for  cancer  research  and  other  laboratory  purposes. 

27  Salmon  and  Smith,  Rep.  of  Com.  of  Agri.,  Wash.,  1885  and  1886. 


692  PATHOGENIC   MICROORGANISMS 

The  fact  that  this  organism  cannot  with  regularity  be  distinguished  from 
the  hog  cholera  bacillus  and  some  other  members  of  the  paratyphoid 
"B"  group,  opens  the  question  as  to  whether  human  beings  can  be 
infected  by  organisms  derived  from  the  disease  in  mice.  In  connection 
with  attempts  at  the  wholesale  destruction  of  mice  by  infecting  bait  in 
traps  with  cultures,  in  the  hope  of  starting  an  epidemic  among  them, 
human  infections  have  been  reported.  Troomsdorf,28  was  the  first  to 
publish  such  cases.  Meyer  29  reported  an  accidental  laboratory  infec- 
tion from  which  he  concluded  that  in  man  the  disease  could  produce  an 
acute  and  rather  severe,  but  short-lived  disease.  Shibajama30  has 
reported  a  number  of  cases  which  he  carefully  investigated.  In  all  of 
them  there  was  definite  circumstantial  evidence  that  the  individuals 
had  been  exposed  to  infection  with  these  organisms.  In  one  of  them 
food  had  been  taken  from  a  wooden  dish  in  which  mouse  typhoid  bacilli 
for  the  infection  for  bait  had  been  kept.  In  another,  a  peasant  woman 
accidentally  mixed  the  mouse  typhoid  cultures  with  flour.  In  another, 
again,  a  number  of  people  had  eaten  meat  of  a  horse  which  had  been 
fatally  infected  by  accidental  mixture  with  its  food  of  mouse  typhoid 
virus.  In  the  last  case,  34  people  were  infected,  one  of  whom  died. 
The  symptoms  were  violent  gastroenteritis,  coming  on  within  twenty- 
four  hours  after  eating  of  the  meat,  and  in  many  respects  similar  to  the 
typical  disease  described  by  Gaertner. 

B.  PESTIS  CAVI^E. — The  guinea  pig  disease  caused  by  the  B.  Pestis 
Cayise  usually  takes  the  form  of  what  is  commonly  known  as  pseudo- 
tuberculosis.  It  may  occur  epidemically  in  laboratory  guinea  pigs  and 
kill  large  numbers. 

DANYSZ  TYPE. — A  definite  group  of  the  organisms,  the  so-called  Ratin 
or  Danysz  group,  produce  epidemic  diseases  among  rats.  By  German 
authors  this  Danysz  group,  described  by  the  worker  whose  name  they 
bear  in  1900,  are  generally  regarded  as  very  close  to  the  true  Gaertner 
bacillus.  It  is  pathogenic  for  guinea  pigs  and  mice,  and  can  be  trans- 
mitted to  rats,  as  well  as  to  these  animals  by  feeding.  There  is,  as  in 
all  other  diseases  of  rodents  caused  by  these  organisms,  a  very  much 
enlarged  spleen,  with  inflammatory  changes  in  the  intestinal  mucous 
membrane  and  necrotic  foci  in  the  organs. 

The  B.  Abortus  Equi,  first  described  by  Smith  and  Kilborne,  has 
been  found  to  be  the  cause  of  infectious  abortion  in  horses,  and  deserves 
more  detailed  description  as  follows : 

28  Troomsdorf,  Munch,  med.  Woch.,  48,  1903. 

29  Meyer,  Munch,  med.  Woch.,  47,  1905. 

30  Shibajama,  Munch  med.  Woch.,  54,  1907,  979. 


BACILLI   OF   THE    COLON-TYPHOID-DYSENTERY    GROUP     693 

B.  ABORTUS  EQUI  AND  INFECTIOUS  ABORTION  OF  HORSES.— The  infec- 
tious abortion  of  horses  and  cattle  has  interested  veterinary  bacteriolog- 
ical workers  for  a  great  many  years,  since,  in  both  cases,  the  diseases  have 
been  the  cause  of  much  economic  loss.  There  is,  however,  considerable 
confusion  in  the  minds  of  general  bacteriological  workers  concerning 
the  various  organisms  that  have  been  described  in  connection  with  these 
diseases  and  there  seem  to  be  two  very  distinct  types  associated  with  the 
condition  in  different  animals,  one  of  them  belonging  to  the  paratyphoid 
or  hog  cholera  group,  the  other  apparently  having  nothing  to  do  with 
these  organisms,  biologically.  We  refer  to  them  both  in  this  place, 
however,  because  anyone  looking  up  the  B.  Abortus  in  this  book  would 
desire  to  have  a  definite  statement  concerning  the  status  of  the  bac- 
teriology of  the  diseases  in  general.  The  idea  that  abortion  of  domestic 
animals  was  an  infectious  disease  really  originated  with  breeders  and  was 
first  suggested  to  scientific  workers  by  Flandrin  in  1806.31  Nothing 
positive  from  the  bacteriological  point  of  view  came  out  of  this  until 
1897  when  Bang32  described  an  organism  which  he  isolated  from  cows 
which  had  aborted.  There  seems  to  be  little  question  about  the 
ability  of  Bang's  organism  to  cause  this  disease  in  cattle.  The 
description  of  the  Bang  organism  which  is  generally  spoken  of  as 
B.  Abortus  Bovis  will  be  found  in  another  part  of  this  book. 

Soon  after  Bang's  announcement,  a  British  Commission  investi- 
gated the  same  subject  with  a  view  of  finding  out  whether  the  abortion 
on  horse  farms  in  England  were  due  to  the  same  bacillus,  but  they 
could  not  confirm  this.  Ostertag33  also  investigated  the  same  subject 
in  1900  in  Germany,  but  could  not  confirm  the  original  suggestion  of 
Bang  that  his  bacillus  caused  abortion  in  horses  as  well  as  in  cattle. 
In  1903,  Smith  and  Kilborne  34  described  a  bacillus  belonging  definitely 
to  the  hog-cholera  group  which  they  had  isolated  from  the  vagina  of 
horses  that  had  aborted,  and  which  they  held  responsible  for  the  disease. 
De  Jong  35  confirmed  these  findings  and  was  able  to  produce  the  disease 
in  mares  by  intravenous  injection.  Numerous  other  investigations 
have  been  made  since  then,  a  recent  one  by  Gminder  36  confirming  every- 
thing claimed  by  Smith  and  Kilborne  in  the  beginning.  Gminder  isolated 

31  Flandrin,  quoted  from  Gmmder,  Arb.  a.  d.  Reichsgesundheitsamte,  52,  1920, 
113. 

32  Bang,  Zcit.  f.  Tier.  Med.,  1,  1897,  241. 

33  Ostertag,  quoted  from  Gminder,  loo.  fit. 

34  Smith  and  Kilborne,  Bull.  No.  3,  IT.  S.  Bur.  Animal.  Ind.,  U.  S.'Treas. 

35  De  Jong,  Cent,  f .  Bakt.,  67,  1912,  148. 

36  Gminder,  Arb.  a  d.  Reichsgesundheitsamte,  52,  1920,  113. 


694  PATHOGEN  1C   MICROORGANISMS 

similar  organisms  from  aborting  mares.  He  showed  specific  agglutinins 
and  complement  fixing  bodies  in  the  sera  of  these  mares  and  produced 
abortion  in  laboratory  animals,  guinea  pigs,  rabbits  and  rats  by  intra- 
venous injection  and  by  feeding. 

Gminder  found  the  Smith-Kilborne  bacillus  most  frequently,  but 
also  found  organisms  closer  to  the  Gaertner  enteritidis  organisms,  and 
others  closely  related  to  the  paratyphoid  "B"  strains,  and  believes 
that  abortion  may  be  due  to  a  number  of  other  closely  related  organisms 
of  the  paratyphoid-en teritidis  groups. 

The  B.  Psittacosis  belonging  to  this  group  was  described  by  Nocard37 
as  the  causative  agent  in  a  disease  of  parrots  and  is  fatal  for  birds  of 
many  different  species.  Nocard  succeeded  in  transferring  it  from  one 
parrot  to  another  by  feeding. 

Durham  38  studied  this  organism  and  pointed  out  its  close  similarity 
to  the  Enteritidis  organisms.  The  possibility  of  infection  in  man  with 
this  organism  was  mentioned  by  Nocard,  and,  though  this  question  can- 
not be  regarded  as  definitely  settled  at  the  present  time,  other  apparent 
infections  from  parrot  to  man  have  been  reported.  Jackson  has 
recently  reported  a  small  epidemic  in  a  Pennsylvania  town  which  he 
thought  he  could  trace,  by  epidemiological  study,  to  original  infection 
from  birds. 

Organisms  of  this  class  have  been  regarded  as  causing  disease  in 
calves  and  cows  and  it  has  been  shown  that  some  of  the  bacteria  men- 
tioned above  could  cause  diseases  in  cattle  and  horses  by  feeding  experi- 
ments. 

In  judging  of  these  animal  diseases, it  must  not  be  forgotten  that 
organisms  belonging  unquestionably  to  the  paratyphoid  group  have 
often  been  found  in  the  intestinal  canals  of  normal  calves,  pigs  and,  in  a 
few  cases,  in  horses,  and  that  it  is  not  unlikely  that  the  bacillus  is  pretty 
generally  distributed  in  the  animal  kingdom  as  perhaps  a  normal  or 
occasional  inhabitant  of  the  intestinal  canal. 

BACILLUS  OF  FOWL  TYPHOID  (B.  SANGUINARIUM,  MOORE). — This 
organism,  together  with  the  B.  Pullorum,  has  been  difficult  to  classify  be- 
cause of  several  definite  and  apparently  fundamental  differences  between 
it  and  other  members  of  the  group.  It  was  isolated  first  by  Moore3' 
from  diseased  fowl.  The  chief  pathological  condition  in  the  fowl  seems 


37  Nocard,  cited  frorm  Uhlenhuth  and  Hubener,  Kolle  and  Wassermann  Handb., 
2d  Edit.  Vol.  3. 

38  Durham,  Brit.  Med.  Jour.,  1898. 

39  Moore.  12th  Annual  Rep.  Bur.  Animal  Industry,  1895. 


BACILLI    OF    THE    COLON-TYPHOID-DYSENTERY    GROUP     695 

to  consist  of  severe  blood  changes  (anaemia),  and  it  was  spoken  -of,  for 
some  time,  as  Infectious  Leukaemia  of  fowls.  The  organism  is  a 
Gram-negative  bacillus,  non-sporulating  and  uncapsulated,  which 
differs  from  other  members  of  the  paratyphoid  group  chiefly  in  being 
non-motile  and  in  not  producing  gas  from  dextrose.  Smith  isolated  a 
similar  organism  from  a  diseased  chicken  in  Rhode  Island  in  1894. 
The  organism  has  been  studied  since  then,  particularly  by  Smith  and 
Ten  Broeck,40  by  Rettger  and  Koser,41  and  by  Krumwiede  and  Kohn.42 
To  summarize,  then,  it  is  a  short,  Gram-negative  bacillus  which  does  not 
form  spores,  and  does  not  liquefy  gelatin.  It  does  not  produce  gas  in 
any  of  the  carbohydrate  media,  but  produces  acid  on  glucose,  mannite, 
maltose  and  xylose.  It  does  not  produce  indol.  An  important  point 
is  that  it  produces  acid  upon  rhamnose,  an  observation  made  by  Krum- 
wiede and  Kohn,  and  this  rhamnose  reaction  is  one  which  this  organism 
has  in  common  with  all  other  organisms  of  the  paratyphoid-enteritidis 
group.  Krumwiede  and  Kohn  regard  the  rhamnose  fermentation  as  an 
essential  characteristic,  differentiating  both  the  aerogenic  and  anaero- 
genic  members  of  this  group  from  B.  Typhosus.  Smith  and  Ten 
Broeck  first  pointed  out  the  close  relationship  of  fowl  typhoid  to  the 
group  in  which  we  are  placing  it,  and  showed  that  it  was  closely  related 
to  the  typhoid  bacilli  by  agglutination  and  agglutinin  absorption. 

B.  PULLORUM  (RETTGER43)  BACILLUS  OF  WHITE  DlARRHEA  IN  FOWLS. 

—This  organism,  like  the  one  preceding  it,  has  a  very  close  antigenic 
relationship  to  B.  Typhosus.  Unlike  B.  Sanguinarium,  it  produces  a 
small  amount  of  gas  in  dextrose  and  mannite,  though  as  Smith  and 
Ten  Broeck  have  shown,  the  gas  producing  property  may  be  sup- 
pressed in  certain  strains,  and  again  resumed  on  further  cultivation. 
A  non-gas  producing  strain  forwarded  by  them  to  Krumwiede  resumed 
its  ability  to  produce  gas  subsequently.  Like  the  preceding,  it  is 
non-motile,  but  B.  Pullorum  produces  no  visible  changes  in  media 
containing  maltose,  dulcite  and  dextrin,  while  B.  Sanguinarium  produces 
acid  in  such  media. 

The  B.  Pullorum  is  the  cause  of  a  spontaneous  epidemic  disease  in 
young  chicks.  It  is  important  to  note,  also,  that  B.  Pullorum  like  the 
B.  Sanguinarium  and  other  members  of  the  paratyphoid  group  produces 
acid  on  rhamnose  (Krumwiede  and  Kohn). 

40  Smith  and  Ten  Broeck,  Jour.  Med.  Res.,  31,  1915,  503  and  523. 

41  Rettger  and  Koser,  Jour.  Med.  Res.,  35,  1916-1917,  443. 

42  Krumwiede  and  Kohn,  Jour.  Med.  Res.,  36,  1917,  509. 

43  Rettger,  Jour.  Exper.  Med.,  19,  1914. 


696  PATHOGENIC  MICROORGANISMS 

PATHOGENICITY  OF  THE  PARATYPHOID  GROUP  FOR  MAN 

Organisms  of  this  class  may  be  the  causative  agents  of  a  number  of 
clinically  varying  conditions  of  man.  In  general,  it  may  be  said  that 
two  main  types  of  disease  can  be  caused  by  this  group,  1,  that  in 
which  the  disease  simulates  a  mild  or  severe  typhoid  fever  recognizable 
as  different  from  true  typhoid  fever  only  by  isolation  and  identification 
of  the  paratyphoid  organisms;  and  2,  those  which  fall  into  the  category 
of  "meat  poisoning"  in  which,  after  a  very  short  incubation  time,  one 
to  two  days  or  even  less,  there  are  symptoms  of  gastroenteritis  which 
may  be  mild,  but  more  frequently  are  explosive  and  severe. 

1.  True  paratyphoid  fever,  or  a  typhoid-like  fever,  may  be  caused 
by  members  of  the  paratyphoid  "A"  or  "B"  group. 

The  paratyphoid  "A"  group  may  be  regarded  as  standing  somewhat 
apart  from  the  other  microorganisms  of  this  large  and  heterogeneous 
family  in  being  quite  sharply  distinguishable  from  the  others,  both  by 
cultural  and  agglutinative  reactions.  The  organism,  unlike  other  para- 
typhoid and  enteritidis  bacilli,  fails  to  ferment  xylose,  as  shown  by 
Krumwiede  and  his  coworkers,  and  fails  to  give  a  positive  reaction  on 
the  lead  acetate  media.  By  agglutination  reactions  the  organisms  of  the 
"A"  type  are  found  to  be  more  homologous  than  others  and  a  power- 
ful "A"  serum  usually  agglutinates  "A"  organisms  in  general,  a  con- 
dition which  is  quite  unlike  that  existing  among  the  "B"  and  other 
members  of  the  group.  Paratyphoid  "A"  is  probably  conveyed  in 
exactly  the  same  way  as  is  typhoid  fever,  namely,  by  water,  milk  and 
direct  and  indirect  contact,  food  contamination,  carriers  and  the  agency 
of  flies. 

The  disease  itself  may  take  the  form  either  of  a  very  mild  and  short- 
lived enteric  disturbance  with  slight  fever,  or  it  may  take  the  form  of  a 
moderately  severe  typhoid  fever  with  rose  spots,  enlargement  of  the 
spleen  and  positive  blood  culture.  Paratyphoid  "A"  cases  are  not  a 
very  large  percentage  of  the  ordinary  sick  rate  of  communities,  but 
occasionally  small  group  epidemics  have  been  studied;  and  during 
recent  years  in  the  United  States,  military  epidemics  have  occurred. 
Upon  the  recent  return  of  some  American  militia  regiments  from 
the  Mexican  Border,  there  appeared  among  them  an  epidemic  of  para- 
typhoid which  consisted  largely  of  the  "A"  type.  We  had  the  privilege 
of  studying  a  good  many  of  these  cases,  and  found  a  most  varied  clinical 
picture.  The  severe  cases  were  practically  indistinguishable  from 
typhoid  fever,  rjut  there  were  at  the  same  time  cases  of  very  mild  fever 
with  nothing  but  a  little  diarrhea,  in  which  diagnosis  was  made  only  by 


BACILLI   OF   THE   COLON-TYPHOID-DYSENTERY   GROUP     G97 

stool  culture  and  in  a  few  cases  by  blood  culture.  A  large  number  of 
the  men  of  these  regiments  turned  out  to  be  paratyphoid  "A"  carriers. 

Paratyphoid  "B"  is  probably  a  more  common  disease  than  para- 
typhoid "A,"  and  is  more  apt  to  be  typhoid-like  and  severe.  From  the 
very  distinct  differences  between  the  clinical  manifestations  of  this  dis- 
ease and  the  ordinary  case  of  so-called  meat  poisoning,  it  would  appear 
that  there  must  be  a  very  definite  human  paratyphoid  "B"  organism 
which  is  conveyed  by  the  same  agencies  and  subject  to  the  same  epidem- 
iological  laws  as  typhoid  fever.  It  is  difficult  to  base  this  on  bac- 
teriological evidence  since  it  is  often  impossible  to  find  any  cultural 
or  agglutinative  distinctions  between  organisms  isolated  from  the 
human  blood  or  bowel,  and  other  bacilli  which,  from  their  sources  and 
general  reactions,  would  fall  into  the  groups  of  hog  cholera,  enteritidis, 
etc.  Numerous  attempts  have  been  made  to  classify  these  organisms 
according  to  source;  one  of  the  last  attempts  to  correlate  group  with 
host-origin  being  that  made  by  Krumwiede,  Pratt  and  Kohn.44  Their 
results  seem  to  indicate  that  reduction  of  fuchsin  and  quantitative  dif- 
ferences in  the  fermentation  of  dulcite  and  arabinose  may  to  some  extent 
bring  about  a  tentative  correlation.  But  it  cannot  be  said  in  any  sense 
at  the  present  time  that  we  can  sharply  differentiate  between '  those 
"B  "'  types  which  invade  human  beings  and  produce  the  typical  typhoid- 
like  disease,  and  those  which  may  originate  in  a  disease  of  animals  and 
be  secondarily  transferred  to  man. 

Paratyphoid  "B,"  then,  occurs  in  man  in  exactly  the  same  way  and 
by  the  same  agencies  as  typhoid  fever,  and  produces  a  disease  indis- 
tinguishable from  typhoid,  except  by  bacteriological  methods. 

In  contradistinction  to  true  typhoid  the  temperature  reaction  of  this 
case  may  set  in  more  abruptly  and  remain  more  irregular  throughout 
the  disease.  Gastric  symptoms,  vomiting,  and  nausea  are  often  more 
prominent  than  in  typhoid  fever  and  enlargement  of  the  spleen  is  less 
regularly  present.  Owing  to  the  low  mortality  of  paratyphoid  fever  (in 
120  cases  observed  by  Lentz  45  less  than  4  per  cent,  and  in  many  other 
smaller  epidemics  no  deaths  have  occurred),  we  have  remained  relatively 
ignorant  concerning  the  pathologic  anatomy  of  the  disease.  Long- 
cope  4G  observed  a  case,  fatal  after  two  weeks  of  illness,  in  which  there 
was  no  enlargement  of  Peyer's  patches  and  no  sign  of  even  beginning 
ulceration.  Most  other  observers  have  also  found  less  involvement  of 

44  To  be  supplied. 

45  Lentz,  Klin.  Jahrb.,  xiv,  1914. 

46  Longcope,  Amer.  Jour,  of  Med.  Sciences,  cxxiv,  1902. 


698  PATHOGENIC   MICROORGANISMS 

the  lymphatics  of  the  bowel  than  is  found  in  typhoid  fever.  During 
the  disease  the  bacteria  can  often  be  cultivated  from  the  blood,  and  the 
serum  of  the  patient  may  agglutinate  specifically  paratyphoid  strains. 
In  this  way  the  diagiiosis  can  often  be  made.  Libmann  47  has  isolated 
the  organism  from  the  fluid  aspirated  from  the  gall  bladder  in  a  case 
operated  on  for  cholecystitis. 

MEAT  POISONING. — As  stated  in  the  beginning  of  this  chapter,  this 
disease  and  its  etiological  causation  were  first  described  by  Gaertner  in 
1888.  His  observations  were  based  upon  an  epidemic  occurring  in 
Germany  in  which  57  cases  of  more  or  less  violent  gastroenteritis 
occurred  in  a  group  of  people  who  had  eaten  the  meat  of  a  condemned 
cow.  From  a  fatal  case  and  from  the  meat  itself,  Gaertner  isolated 
the  organisms  which  bear  his  name. 

Since  then,  many  similar  observations  have  been  made.  Uhlenhuth 
and  Hiibener  28  have  made  a  very  thorough  study  of  the  literature,  to 
which  the  reader  is  referred  for  more  extensive  treatment  of  the  subject. 
They  have  tabulated  a  large  number  of  outbreaks  which  took  place  in 
Germany  and  the  neighboring  countries  between  the  years  1885  and 
1910.  A  considerable  number  of  these  were  studied  by  competent 
bacteriologists  and  can  be  regarded  as  reliably  reported.  It  appears 
that  beef  and  pork  are  the  most  common  sources  of  infection,  whereas 
infection  is  also  possible  from  horse  meat  and  mutton.  In  the  majority 
of  cases  the  disease  followed  the  ingestion  of  the  meat  of  cattle  that  were 
diseased  before  being  slaughtered.  The  fact  that  the  organisms  have 
appeared  in  the  circulation  of  the  animal  before  death  and  have  perhaps 
accumulated  in  the  meat  that  is  eaten,  may  account  for  the  acute  nature 
of  the  disease  and  the  severe  toxic  symptoms,  since,  as  we  know,  the 
poisons  of  the  bacterial  bodies  of  organisms  of  this  group  may  be  quite 
powerful,  may  act  directly  upon  the  gastro-intestinal  mucosa  and  may 
not  be  destroyed  by  considerable  heating.  It  must  not  be  forgotten, 
however,  that  organisms  belonging  to  the  paratyphoid  "  B  "  and 
enteritidis  groups  may  be  present  in  the  intestinal  canals  of  a  large 
percentage  of  normal  animals  of  the  species  mentioned  and  that  to  some 
extent  invasion  of  the  organs  may  be  a  post-mortem  phenomenon. 
The  studies  of  most  observers,  however,  seem  to  indicate  that  the  most 
severe  infections  occur  when  the  animal  was  diseased  before  .being  killed. 
Again,  there  have  been  cases  in  which  infection  has  been  due  to  second- 


47  Libmann,  Jour,  of  Med.  Res.,  viii,  1902. 

48  Uhlenhuth  and  Hiibener ,  Kolle  and  Wassermann  Handb.,  2d  edit.  Vol.  3. 


BACILLI   OF   THE   COLON-TYPHOID-DYSENTERY   GROUP     699 

ary  contamination  of  the  meat  either  by  a  carrier  or  by  another  agency 
exactly  as  this  may  occur  in  typhoid  transmission. 

The  clinical  picture  of  this  variety  of  paratyphoid  infection  is  funda- 
mentally different  from  the  one  just  described.  Topical  bacillary  meat 
poisoning  comes  on  within  less  than  twenty-four  hours  after  the  ingestion 
of  the  meat.  Its  onset  is  acute  with  a  rapid  rise  in  temperature  and  the 
accompanying  systemic  symptoms  which  this  implies.  There  is  usually 
great  prostration,  rapidity  of  the  pulse  and  localized  symptoms  referable 
to  the  gastro-intestinal  canal,  namely,  nausea,  vomiting  and  painful 
diarrhea.  Some  of  these  cases  have  been  compared  with  cholera  in  the 
severity  of  their  courses.  The  mortality  has  not  usually  been  very  high, 
although  in  one  epidemic  it  was  as  high  as  7  per  cent.  The  organisms 
may  in  these  cases  also  be  found  in  the  blood  stream  by  culture,  but  not 
as  regularly  as  in  the  typical  typhoid  fever-like  form.  At  death  they 
may  be  found  in  the  spleen  and  intestines. 

From  the  epidemiological  point  of  view,  it  is  always  important,  when 
a  group  of  persons  after  a  traceable  common  meal  is  seized  with  acute 
gastroenteritis,  to  make  an  epidemiological  survey,  study  the  time  and 
place  of  the  common  meal,  follow  up  the-  subsequent  histories  of  others 
who  were  present  at  this  meal,  and,  if  possible,  secure  for  bacteriological 
study  some  of  the  meat,  milk,  etc.,  consumed. 

Preventive  Measures. — Measures  for  the  prevention  of  the  typhoid- 
like  forms  of  paratyphoid  fever  are  identical  with  those  advised  for  the 
prevention  of  typhoid.  The  carrier  problem  is  practically  the  same  and 
it  seems  logical  to  assume  that  the  percentage  of  carriers  compared  with 
that  of  typhoid  carriers  is  approximately  similar  to  the  ratio  of  incidence 
between  the  two  diseases. 

In  regard  to  the  meat  poisoning  epidemics,  preventive  measures 
must  be  chiefly  aimed  at  the  sanitary  control  of  slaughter  houses  and 
care  in  food  preparation.  Concerning  the  slaughter  house  survey,  this 
must  go  farther  than  simply  controlling  the  health  of  animals  before 
slaughter,  since,  as  we  have  seen  above,  many  organisms  of  these  types 
may  be  found  in  the  normal  intestines  of  animals.  Though  studies  in 
this  direction  have  not  been  made  with  sufficient  extensiveness,  it  is  still 
suggested  by  our  general  knowledge  of  this  subject  that  an  unduly  long 
interval  between  evisceration  and  slaughter,  especially  in  warm  weather, 
may  lead  to  invasion  and  multiplication,  in  the  tissues,  of  organisms 
from  the  bowels,  which  may  render  the  meat  of  a  previously  healthy 
animal,  unsafe.  Removal  of  the  intestines  promptly  after  slaughter 
would  be  the  most  important  preventive  measure  that  reasoning  would 
indicate. 


'      CHAPTER  XXXIV 

BACILLI   OF   THE   COLON-TYPHOID-DYSENTERY   GROUP  (Continued} 
BACILLARY  DYSENTERY  AND  THE  DYSENTERY  BACILLI 

ALTHOUGH  acute  dysentery  has  been  an  extremely  prevalent  disease, 
occurring  almost  annually  in  epidemic  form  in  some  of  the  Eastern  coun- 
tries and  appearing  sporadically  all  over  the  world,  its  etiology  was 
obscure  until  1898  when  Shiga  1  described  a  bacillus  which  he  isolated 
from  the  stools  of  patients  suffering  from  this  disease  in  Japan,  and 
established  with  scientific  accuracy  its  etiological  significance.  Since  the 
discovery  of  Shiga's  bacillus  a  number  of  other  bacilli  have  been 
described  by  various  workers,  all  of  which,  while  showing  slight  biological 
differences  from  Shiga's  microorganism,  are  sufficiently  similar  to  it 
culturally  and  pathogenically  -to  warrant  their  being  classified  together 
with  it  in  a  definite  group  under  the  heading  of  the  "  dysentery  bacilli." 

The  manner  in  which  Shiga  made  his  discovery  furnishes  an  instruct- 
ive example  of  the  successful  application  of  modern  bacteriological 
methods  of  etiological  investigation.  Many  workers  preceding  Shiga 
had  attempted  to  throw  light  upon  this  subject  by  isolations  of  bacilli 
from  dysenteric  stools,  and  by  extensive  animal  inoculation.  Shiga, 
following  a  suggestion  made  by  Kitasato,  approached  the  problem  by 
searching  for  a  microorganism  in  the  stools  of  dysentery  patients  which 
would  specifically  aggrutinate  with  the  serum  of  these  patients.  His 
labors  were  crowned  with  success  in  that  he  found,  in  thirty-six  cases, 
one  and  the  same  microorganism  which  showed  uniform  serum  agglu- 
tinations. Further,  he  found  that  this  bacillus  was  not  present  in  the 
dejecta  of  patients  suffering  from  other  diseases  nor  in  those  of  normal 
men,  and  that  when  tested  against  the  blood  serum  of  such  people  it 
was  not  agglutinated. 

Description  of  Shiga's  Bacillus. — Shiga's  bacillus  is  a  short  rod,  rounded 
at  the  ends,  morphologically  very  similar  to  the  typhoid  bacillus,  and,  like  it, 
inclined  to  involution  forms.  The  organism  generally  occurs  singly,  more  sel- 
dom in  pairs.  It  is  decolorized  by  Gram's  method  of  staining.  With  the 

1  Shiga,  Cent.  f.  Bakt.,  xxiii,  1898;  ibid.,  xxiv,  1898;  Deut.  med.  Woch.,  xliii, 
xliv,  and  xlv,  1901. 

700 


BACILLI   OF   THE   COLON-TYPHOID-DYSENTERY    GROUP     701 

ordinary  anilin  dyes  it  stains  easily,  showing  a  tendency  to  stain  with  slightly 
greater  intensity  at  the  ends.  The  organism  is  an  aerobe  and  facultative  anaer- 
obe. Although  described  at  first  by  Shiga  as  being  motile,  its  motility  has  not 
been  satisfactorily  proven,  and  most  observers  agree  in  denying  the  presence  of 
flagelte,  and  affirming  the  complete  absence  of  motility. 

On  cigar  the  colonies  are  not  characteristic,  resembling  those  of  the  typhoid 
bacillus. 

On  gelatin,  the  colonies  appear  very  much  like  typhoid  colonies  and  the  gela- 
tin is  not  liquefied.  On  potato,  the  growth,  like  that  of  typhoid,  is  at  first  not 
visible,  but  after  about  a  week  turns  reddish  brown. 

In  broth,  there  is  clouding,  with  moderate  deposits  after  some  days.  No 
pellicle  is  formed. 

Milk  is  not  coagulated.  Litmus  milk  shows  a  slight  primary  acidity,  later 
again  becoming  alkaline  and  taking  on  a  progressively  deeper  blue  color. 

Indol  is  not  formed. 

No  gas  is  formed  in  media  containing  dextrose,  lactose,  saccharose,  or  other 
carbohydrate. 

While  not  delicately  susceptible  to  reaction,  the  bacillus  prefers  slightly 
alkaline  media. 

Shiga  differentiated  his  organism  from  the  typhoid  bacillus  chiefly  by  sup- 
posed differences  in  colony  characters  and  by  the  agglutination  reaction. 

Following  the  work  of  Shiga,  a  large  number  of  investigators  turned 
their  attention  to  the  subject  of  dysentery,  with  the  result  that  many 
new  forms  were  discovered  and  at  first  a  considerable  amount  of  con- 
fusion prevailed. 

Flexner 2  in  1899  investigated  dysentery  in  the  Philippines,  and 
isolated  a  bacillus  which,  he  considered,  corresponded  to  Shiga's 
organism. 

Strong  and  Musgrave3  in  1900  described  a  bacillus  isolated  from 
dysentery  cases  in  the  Philippines  which  was  essentially  like  that  of 
Flexner. 

Nearly  simultaneously  with  the  papers  of  Flexner  and  of  Strong  and 
Musgrave,  Kruse  4  published  investigations  of  an  epidemic  of  dysentery 
occurring  in  Germany.  His  observations  were  of  the  greatest  impor- 
tance and  largely  formed  the  starting  point  of  the  further  advances 
which  have  been  made  in  the  etiology  of  dysentery. 

Kruse's  organism  was  described  as  forming  colonies  on  gelatin  and 
agar,  practically  like  those  of  Bacillus  typhosus.  Like  this  bacillus,  no 
gas  was  formed  from  grape  sugar,  and  the  growth  in  milk  and  on  potato, 

2  Flexner,  Phila.  Med.  Jour.,  vi,  1900,  and  Bull.  Johns  Hopkins  Hosp.,  xi,  1900. 

3  Strong  and  Musgrave,  Report  Surg.  Gen.  of  Army,  Washington,  1900. 

4  Kruse,  Deut.  med.  Woch.;  xxvi,  1900. 


702  PATHOGiEilC   MICROORGANISMS 

and  even  in  Piorkowski's  urine  gelatin,  resembled  that  of  Bacillus 
typhosus.  According  to  Kruse,  this  organism  was  absolutely  without 
motility. 

In  1901  Kruse5  contributed  a  second  paper.  In  this,  besides  con- 
firming his  previous  observations,  he  described  another  class  of  organ- 
ism coming  from  cases  which  he  designated  as  "pseudo-dysentery  of 
insane  asylums."  In  the  case  of  one  patient,  and  at  two  autopsies,  he 
isolated  organisms  which  he  could  not  distinguish,  morphologically  or 
culturally,  from  the  true  dysentery  bacillus,  but  which  showed  differ- 
ences in  their  serum  reaction.  By  careful  study  of  the  behavior  of  these 
bacilli  in  the  serum  of  patients  and  in  immune  serum  from  animals  he 
not  only  showed  that  they  were  different  from  the  original  cultures 
from  cases  of  epidemic  dysentery  which,  no  matter  what  their  source, 
were  found  to  be  alike,  but  that  they  showed  differences  among  them- 
selves and  apparently  fell  into  two  or  more  varieties.  One  of  these 
organisms  culturally  and  by  its  serum  reactions  showed  itself  practically 
identical  with  one  of  the  cultures  he  had  received  from  Flexner. 

Spronck  6  in  1901  described  an  organism  isolated  in  Utrecht  from 
dysentery  cases,  which  showed  great  similarity  to  the  Shiga-Kruse 
organism;  but,  when  tested  in  the  serum  of  a  horse  immunized  against 
true  dysentery  bacillus,  showed  practically  no  agglutination.  He 
placed  this  organism  in  the  group  designated  by  Kruse  as  the  "  pseudo- 
dysentery  bacilli."  His  communication  is  of  importance,  since  it  is  the 
first  reported  instance  in  which  any  investigator  had  recognized  and 
associated  the  so-called  pseudo-dysentery  bacilli  with  dysentery  ap- 
proaching the  acute  epidemic  form  in  type. 

Following  this  work  a  number  of  investigators,  including  Vedder 
and  Duval,7  Flexner,  and  Shiga  8  himself,  published  communications  in 
which  they  claimed  identity  for  the  various  forms  previously  described. 

In  1902  Park  9  and  Dunham  described  an  organism  which  they 
found  in  a  small  outbreak  of  dysentery  occurring  in  Maine.  This 
organism  differed  from  most  of  those  previously  described  in  that  it 
was  found  to  produce  indol  in  pepton  solutions. 

In  the  same  year  Martini 10  and  Lentz  published  an  article  in  which 
they  attempted  to  differentiate  various  dysentery  bacilli  by  means  of 

5  Kruse,  Deut.  med.  Woch.,  xxvii,  1901. 

6  Spronck,  Ref.  Baumgarten's  Jahresber.,  1901. 

7  Vedder  and  Duval,  Jour.  Exp.  Med.,  vi,  1902. 
6  Shiga,  Zeit.  f.  Hyg.,  41,  1902. 

9  Park  and  Dunham,  N.  Y.  Univ.  Bull,  of  Med.  Sci.,  1902. 
1°  Martini  und  Lentz,  Zeit.  f.  Hyg.:  xli,  1902. 


BACILLI    OF    THE    COLON-TYPHOID-DYSENTERY    GROUP  .  703 

agglutination.  This  research  is  of  importance  in  that  it  supported  the 
work  of  Kruse  and  of  Spronck,  indicating  a  difference  between  the 
agglutinative  character  of  the  Kruse  organism  and  the  so-called  "  pseudo- 
dysentery"  type,  in  which  Flexner's  organisms  were  included.  It  is  of 
further  interest,  since  it  indicated  a  marked  difference  between  Flexner's 
Philippine  cultures  and  the  Philippine  culture  of  Strong,  the  Strong 
organism  refusing  to  agglutinate  not  only  in  "Shiga"  immune  serum, 
but  also  in  "Flexner"  immune  serum. 

Simultaneously  with  this  article  Lentz  u  published  the  results  of 
comparative  cultural  researches  with  dysentery  and  "  pseudo-dysen- 
tery" bacilli,  in  which  he  made  the  important  observation  that  the 
original  Shiga-Kruse  bacilli  did  not  affect  mannit,  while  the  "  pseudo- 
dysentery"  bacilli,  including  Flexner's  and  Strong's  Philippine  cultures, 
fermented  mannit,  giving  rise  to  a  distinct  acid  reaction  in  the  medium. 
The  Flexner  organisms  and  others  of  the  "  pseudo-dysentery "  bacilli, 
however,  fermented  maltose,  while  the  Shiga-Kruse  type,  as  well  as 
Strong's  bacillus,  left  it  unchanged  at  the  end  of  forty-eight  hours. 

In  January,  1903,  Hiss  and  Russell12  described  a  bacillus  ("Y") 
from  a  case  of  fatal  diarrhea  in  a  child,  which  by  ordinary  cultural  test 
and  absence  of  motility  was  found  to  resemble  the  Shiga-Kruse  and 
Flexner  bacilli.  Immediately  upon  its  isolation,  it  was  found,  how- 
ever, to  differ  from  the  Kruse  culture  by  its  ability  to  ferment  mannit. 
This  observation  was  made  independently  of  Lentz's  work,  which,  at 
that  time,  had  not  become  known  in  America.  In  the  comparative 
study  of  Hiss  and  Russell  on  the  fermentative  abilities  of  various  dysen- 
tery cultures,  the  serum  water  media  (described  on  page  157)  were  used. 
By  the  use  of  these  media,  it  was  found  that  the  Kruse  culture,  a  culture 
of  Flexner's  bacillus  from  the  Philippines,  and  Duval's  "New  Haven" 
culture  fermented  dextrose  with  the  production  of  a  solid  acid  coagu- 
lum,  but  did  not  affect  mannit,  maltose,  saccharose,  or  dextrin.  The 
culture  of  Hiss  and  Russell,  on  the  other  hand,  fermented  not  only 
dextrose  but  also  mannit  with  the  production  of  acid  and  coagulation 
of  the  medium.  Maltose,  saccharose,  and  dextrin  were  not  fermented. 
The  "  Y"  bacillus,  furthermore,  was  shown  to  differ  entirely  from  the  cul- 
tures of  Shiga,  Kruse,  and  "New  Haven"  in  the  serum  of  immunized 
animals.  This  serum  had  for  bacillus  "Y"  a  titer  of  1  :  500  while  the 
three  other  above-named  organisms  did  not  agglutinate  in  it  at  any 
dilution.  In  normal  beef  serum,  the  Hiss-Russell  organism  was  found 


11  Lentz,  Zeit.  f.  Hyg.,  xli,  1902. 

12  Hiss  and  Russell.  Med.  News.  Feb.,  1903. 


704  PATHOGENIC   MICROORGANISMS 

to  agglutinate  as  highly  at  1  :  320,  while  the  other  three  cultures  gave 
no  reaction  in  dilutions  of  over  1  :  10  or  20. 

Park  and  Carey,13  in  March,  1903,  described  an  epidemic  of  dysen- 
tery occurring  in  the  town  of  Tuckahoe,  near  New  York  city,  and 
isolated  an  organism  which  resembled  the  Shiga-Kruse  bacilli  in  not 
fermenting  mannit,  but  produced  indol  in  pepton  solution  after  five 
days.  It  corresponded  in  agglutination  with  the  cultures  "New 
Haven"  and  "Shiga"  when  tested  in  the  serum  of  a  goat  immunized 
against  the  mannit-fermenting  culture  "Baltimore,"  i.e.,  did  not  react 
at  1  :  50,  whereas  Flexner's  "Manila"  and  "Baltimore"  cultures,  Park 
and  Dunham's  "Seal  Harbor"  culture,  and  some  New  York  cultures,  all 
fermenting  mannit,  agglutinated  up  to  two  thousand  dilution  in  the 
"Baltimore"  serum. 

The  preceding  review  of  a  part  of  the  literature,  by  which  our  knowl- 
edge of  the  dysentery  bacilli  was  developed,  demonstrates  sufficiently 
that  we  have  to  deal  in  this  group  with  a  number  of  different  micro- 
organisms. This,  as  we  have  seen,  was  a  fact  first  recognized  by  Kruse 
when  he  spoke  of  his  true  dysentery  and  his  pseudo-dysentery  strains. 
In  spite  of  much  confusion  at  first,  the  careful  study  of  fermentation 
phenomena,  of  specific  agglutinations,  and,  more  recently,  by  Ohno  14 
and  others  of  the  bacteriolytic  phenomena  in  immune  sera,  has  made 
it  possible  to  distinguish  sharply  between  a  number  of  groups. 

Basing  the  grouping  of  these  microorganisms  upon  a  careful  study  of 

fermentations,  Hiss  15  has  divided  them  as  follows: 

* 

"Shiga" 

"Kruse"  \  Ferment  dextrose.     Group  I. 

"New  Haven" 

"Y"  (Hiss  and  Russell  type) 

"Seal  Harbor  "  Ferment  dextrose  and  mannit.     Group  II. 

"Diamond" 

"Ferra" 

''Strong"  (type)  Ferments  dextrose,  mannit,  saccharose.     Group  III. 

rns     (  ypi  Ferment     dextrose,    mannit,    maltose,    saccharose, 

„  dextrin.     Fermentation  of  saccharose  (as  a  rule) 

only  after  six  days.     Group  IV. 
"Wollenstem" 

It  was  noticed,  it  should  be  mentioned,  however,    that   in    the   case   of   the 


13  Park  and  Carey,  Jour.  Med.  Res.,  ix,  1903. 

14  Ohno,  Philippine  Jour,  of  Sci.,  1,  ix.,  1906. 

15  Hiss,  Jour.  Med.  Res.,  N!  S.,  viii,  1904. 


BACILLI   OF   THE   COLON-TYPHOID-DYSENTERY    GROUP     705 

"Y,"  "Diamond,"  and  "Ferra"  there  was  usually  delayed  acid  fermentation  of 
maltose,  never  any  of  dextrin. 

In  studying  the  agglutinative  characters  of  these  groups,  furthermore,  it 
was  found  that  fermentation  tests  and  agglutinations  went  hand  in  hand. 
The  following  table  will  illustrate  the  point; 16 

SERUM  OF  RABBIT  IMMUNIZED  AGAINST  GROUP  I.     (Shiga's  culture). 

Bacilli  of  Group  I.: 

"Shiga  "  (homologous) 20,000 

"Kruse" 20,000 

"New  Haven" 20,000 

Bacilli  of  Group  II.: 

"Y"  200 

"  Ferra  " 200 

"Seal  Harbor" 200 

Bacilli  of  Group  IV.: 

"  Baltimore  " 800 

"Harris" 800 

"Gray" ' 800 

"  Wollstein" 800 

SERUM  OF  RABBIT  IMMUNIZED  AGAINST  GROUP  II.  ("Y"  culture, 
Hiss  and  Russell). 

Bacilli  of  Group  I.: 

"Shiga" lessthan  100 

"Kruse" 100 

"New  Haven" 100 

Bacilli  of  Group  II.: 

"Y"  (homologous) 6,400 

"Ferra" 6,400 

"Seal  Harbor 6,400 

Bacilli  of  Group  IV.: 

"Baltimore" 1,600 

"Gray" 1,600 

"Harris" 1,600 

"Wollstein" 1,600 

SERUM  OF  RABBIT  IMMUNIZED  AGAINST  GROUP  IV.  ("Baltimore" 
culture). 

16  Hiss,  Jour,  of  Med.  Research,  13,  N.  S..  viii,  1904. 


706  PATHOGENIC   MICROORGANISMS 

Bacilli  of  Group  I.: 

"Shiga" .' less  than  100 

"Kruse"..  . 100 

"  New  Haven" 100 

Bacilli  of  Group  II.: 

"Y" 400 

"Ferra" 400 

"Seal  Harbor" 400 

Bacilli  of  Group  IV. : 

"Baltimore"  (homologous) 3,200 

"Harris" 3,200 

"Gray" 3,200 

"  Wollstein" 3,200 

We  have  described  the  above  experiments  of  Hiss  in  considerable 
detail  not  because  we  believe  they  represent  the  final  classification  of 
the  dysentery  group,  but  because  they  illustrate  the  close  antigenic 
relationship  of  the  dysentery  bacilli  and  the  relatively  close  correlation 
between  cultural  and  serological  properties  which  they  revealed. 

There  seems  to  be  little  doubt  about  there  being  a  large  number  of 
different  dysentery  bacilli  which  vary  from  each  other  in  minor  charac- 
teristics, and  it  is  very  difficult  to  be  sure  whether  one  is  dealing  with 
permanent  type  differences  or  with  temporary  variations  or  suppressions. 
For  this  reason-,  it  may  be  best  to  state  definitely  what  the  common 
characteristics  are  which  belong  to  organisms  of  this  group  and  into 
what  main  subdivisions  they  can  be  classified. 

All  the  dysentery  bacilli  are  Gram-negative  bacilli,  usually  slender, 
but  on  occasion,  especially  in  old  cultures,  short  and  plump,  being  mor- 
phologically indistinguishable  with  certainty  from  typhoid  or  similar 
bacilli..  There  are  all  of  them  non-motile  and  non-spore  bearing.  They 
do  not  liquefy  gelatin.  None  of  them  produce  gas  on  carbohydrate 
media,  none  of  them  produce  acid  on  lactose. 

They  grow  easily  on  the  ordinary  media,  their  colonies  on  agar  and 
gelatin  being  like  those  of  typhoid  but  apt  to  be  more  delicate.  On 
broth  they  produce  an  even  clouding  and  on  differential  media,  like 
those  of  Endo,  Conradi-Drigalski,  etc.,  they  grow  like  typhoid  but 
rather  more  delicately. 

The  main  types  into  which  they  can  be  divided  are  the  Shiga  type, 
the  Flexner  type,  the  Bacillus  "Y"  type  of  Hiss  and  Park  and  the 
Strong  type.  By  many  observers,  notably  Park,  the  Shiga  type  is  the 
only  one  that  is  spoken  of  as  the  B.  dysenteric,  the  other  types  being 


BACILLI   OF   THE    COLON-TYPHOID-DYSENTERY   GROUP     707 

spoken  of  as  the  paradysentery  bacilli.     Differentiation  between  the 
four  types  can  be  made  by  sugar  fermentations  as  follows: 


Dextrose 

Mannite 

Maltose 

Saccharose 

B  dvs. 

"Shiga"  

+ 

B.  dys. 
B  dys 

"Flexner"  

«Y" 

+ 
4. 

+ 
-f 

+ 

- 

B.  dys. 

"Strong"  

+ 

+ 

- 

+ 

+  =  acid  (no  gas) . 

The  Shiga  bacillus  also  is  the  only  one  of  these  fixed  types  which  does 
not  produce  indol.  We  do  not  believe,  as  we  did  before,  that  these 
types  represent  all  possible  variants  of  the  dysentery  bacilli.  During 
the  recent  war  epidemics,  we  ourselves,  as  well  as  many  others,  isolated 
non-motile,  Gram-negative  bacilli  from  the  stools  of  mild  dysentaroid 
cases  which  presented  cultural  peculiarities  and  did  not  agglutinate  with 
the  type  sera,  and  many  organisms  have  been  described  by  various 
observers  which  do  not  fit  in  with  any  of  the  main  sub-divisions. 

Resistance  of  Dysentery  Bacilli. — Not  being  spore  bearers,  dysen- 
tery bacilli  are  not  very  resistant  to  heat  and  chemicals.  They  are 
destroyed  at  60°  within  ten  minutes,  and  the  usual  strength  of  the  com- 
mon chemical  disinfectants  kill  them.  Their  resistance  to  the  ordinary 
conditions  in  nature  is,  of  course,  the  important  feature  of  the  epidemi- 
ology of  the  disease.  Accordingly,  a  considerable  amount  of  research 
has  been  done  on  these  problems.  We  quote  the  following  facts  from 
Vincent  and  Muratet.17  In  garden  soil  they  have  been  known  to  live 
from  six  to  fifteen  days,  and  up  to  forty-nine  days  at  a  depth  of  12  inches. 
In  damp  sand  they  have  been  known  to  live  as  long  as  thirty-nine  days. 
Cultures  in  broth  have  lived  for  twenty-five  days,  and  they  have  lived 
more  than  thirty  days  in  dejecta  buried  in  the  soil,  and  on  linen  folded 
up.  In  ice,  they  may  live  for  longer  than  a  month.  Exposure  to  sun- 
light of  course  destroys  them  rapidly.  We  would  like  to  remark  in 
connection  with  the  viability  of  all  organisms  outside  the  animal  body, 
that  such  statements  must  always  be  limited  to  the  peculiar  conditions  of 
symbiosis,  light,  moisture,  temperature',  etc.,  existing  under  the  particular 
conditions  investigated.  If  we  remember  that  non-spore  bearing  organ- 
isms, like  the  typhoid,  the  dysentery  bacilli,  etc.,  can  be  kept  alive  for 
very  long  periods  if  young  agar  growths  are  kept  in  sealed  jars  in  a  cool 
dark  place,  we  may  understand  that  under  peculiarly  favorable  conditions 

17  Vincent  and  Muratet,  Military  Med.  Manual,  Univ.  of  London  Press,  1917, 


708  PATHOGENIC   MICROORGANISMS 

in  nature,  organisms  have  survived  far  beyond  anything  suggested  by 
our  knowledge  of  their  general  resistance  under  adverse  conditions. 

Poisonous  Products  of  the  Dysentery  Bacilli. — The  separate  types 
of  dysentery  bacilli  vary  exceedingly  in  their  powers  to  produce  toxic 
substances.  Of  all  the  various  types  which  have  been  described,  the 
strongest  poisons  have  been  produced  with  bacilli  of  the  Shiga-Kruse 
variety,  less  regularly  active  ones  with  bacilli  of  the  Flexner  and  of  the 
"Y"  type.  In  fact,  investigations  carried  out  with  the  Shiga  bacillus 
have  tended  to  show  that  the  disease  itself  is  probably  a  true  toxemia, 
its  symptoms  being  referable  almost  entirely  to  the  absorption  of  the 
poisonous  products  of  the  bacillus  from  the  intestine. 

The  earliest  investigations,  carried  on  chiefly  upon  rabbits,  which 
are  more  susceptible  to  this  poison  than  any  other  animals,  showed  that 
even  small  doses  of  cultures  of  this  bacillus  administered  intravenously 
or  subcutaneously  would  produce  death  within  a  very  short  time. 
Conradi,18  Vaillard  19  and  Dopter,  and  others,  finding  that  toxic  symp- 
toms were  almost  as  pronounced  when  dead  cultures  were  given  as  when 
the  living  bacilli  were  administered,  came  to  the  conclusion  that  the 
poisons  of  this  bacillus  were  chiefly  of  the  endotoxin  type.  More 
recently  Todd  20  Kraus,21  and  Rosenthal 22  have  claimed  independently 
that  they  were  able  to  demonstrate  strong  soluble  toxins,  similar  in 
every  way  to  diphtheria  toxin.  Kraus  and  Doerr,23  moreover,  claim 
to  have  further  corroborated  this  by  producing  specific  antitoxins  with 
these  substances. 

It  is  easy  to  obtain  poisonous  substances  from  dysentery  cultures 
in  considerable  strength,  both  by  extracting  the  bacilli  themselves  and 
by  nitration  of  properly  prepared  cultures.  It  is  therefore  not  unlikely 
that  both  types  of  poison  are  produced  by  the  bacilli.  Neisser  and 
Shiga24  obtained  toxins  by  emulsifying  agar  cultures  in  sterile  salt 
solution,  killing  the  bacilli  at  60°  C.,  and  allowing  them  to  extract  at 
37.5°  C.  for  three  days  or  more.  The  filtrates  from  such  emulsions 
were  extremely  toxic.  The  simplest  method  of  obtaining  poisons  from 
these  bacilli  is  to  cultivate  them  for  a  week  or  longer  upon  moderately 
alkaline  meat-infusion  broth.  At  the  end  of  this  time,  the  micro- 

18  Conradi,  Deut.  med.  Woch.,  1903. 

19  Vaillard  et  Dopter,  Ann  de  1'inst.  Pasteur,  1903. 

20  Todd,  Brit.  Med.  Jour.,  Dec.,  1903,  and  Jour,  of  Hyg.,  4,  1904. 

21  Kraus,  Monatschr.  f .  Gesundheit,  Suppl.  II,  1904. 

22  Rosenthal,  Deut.  med.  Woch.,  1904. 

23  Kraus  and  Doerr,  Wien.  klin.  Woch.,  xlii,  1905. 

24  Neisser  and  Shiga,  Deut.  med.  Woch.,  1903. 


BACILLI   OF   THE   COLON-TYPHOID-DYSENTERY    GROUP     709 

organisms  themselves  may  be  killed  by  heating  to  60°  and  the  cultures 
filtered.  According  to  Doerr,25  the  toxins  may  be  obtained  in  the  dry 
state  by  precipitation  with  ammonium  sulphate  and  re-solution  of  the 
precipitate  in  water. 

More  recently,  Olitsky  and  Kligler28  have  repeated  and  extended 
the  thorough  study  of  the  Shiga  dysentery  toxin  made  by  Todd.27  These 
writers  differentiate  definitely  between  a  so-called  exotoxin  and  an  endo- 
toxin.  Their  exotoxin  they  obtained  by  growing  the  Shiga  bacilli  for  five 
days  in  alkalin-egg  broth.  Their  endotoxin  was  produced  by  incubating 
agar  growths  in  salt  solution  for  two  days  and  filtering.  The  exotoxin 
in  small  fractions  of  a  cubic  centimeter,  after  an  incubation  time 
of  a  few  hours  to  four  days,  produces  typical  paralysis  and  severe 
nerve  lesions  in  rabbits.  This  poison  was  killed  by  75°  C.  after  one 
hour,  and  powerful  neutralization  was  obtained  with  the  serurn  of 
horses  immunized  with  it.  Their  endotoxin,  so-called,  produces 
loss  of  weight  and  diarrhea  in  the  animals,  but  no  paralysis.  In 
general,  their  results  agree  with  those  of  Krause,  Todd,  Pfeiffer  and 
Ungermann,28  and  Bessau.29  They  have  produced  far  more  potent 
toxins  and  more  powerful  antitoxins  than  previous  workers. 

The  action  of  the  dysentery  toxin  upon  animals  is  extremely  char- 
acteristic and  throws  much  light  upon  the  disease  in  man.  The  injec- 
tion of  a  large  dose  intravenously  into  rabbits  causes  a  rapid  fall 
in  temperature,  marked  respiratory  embarrassment,  and  a  violent 
diarrhea.  This  is  at  first  watery,  later  contains  large  amounts  of  blood. 
If  the  animals  live  a  sufficient  length -of  time,  paralysis  may  occur,  the 
animal  may  fall  to  one  side  or  may  drag  its  posterior  extremities.  It  is 
a  remarkable  fact  that  intravenous  inoculation  gives  rise  to  intestinal 
inflammation  of  a  severe  nature,  unquestionably  due  to  the  excretion 
of  the  poison  by  the  intestinal  mucosa  and  limited,  usually,  to  the 
cecum  and  colon,  rarely  attacking  the  small  intestine.  Flexner,30  who 
has  experimented  extensively  upon  this  question,  believes  it  probable 
that  most  of  the  pathological  lesions  occurring  in  the  intestinal  canal 
of  dysentery  patients  are  referable  to  this  excretion  of  dysentery  toxin, 
rather  than  to  the  direct  local  action  of  the  bacilli. 


25  Doerr,  "Das  Dysenterie toxin,"  Jena,  1907. 

26  Olitsky  and  Kligler,  Jour.  Exper.  Med.,  31,  1920,  19. 
"  Todd^BTit.  Med.  Jour.,  2,  1903,  1456. 

28  Pfeiffer  and  Ungermann,  Cent,  f .  Bakt.,  Orig.,  50,  1909. 

29  Bessau,  Cent.  f.  Back.   57,  1911,  21. 

30  Flexner,  Jour.  Exp    Med.,  8,  1906. 


710  PATHOCJKXIC    MICROORGANISMS 

Characteristic,  of  course,  for  the  Sniga  poison  is  the  paralytic  action 
which  seems  to  be  a  specific  phenomenon  characteristic  of  this  bacillus. 

Immunization  with  Dysentery  Bacilli. — The  immunization  of 
small  animals,  such  as  rabbits  and  guinea-pigs,  against  dysentery  bacilli, 
especially  those  of  the  Shiga  type,  is  attended  with  much  difficulty, 
owing  to  the  great  toxicity  of  the  cultures.  Nevertheless,  successful 
results  may  be  accomplished  by  the  administration  of  extremely  small 
doses  of  living  or  dead  bacilli,  increased  very  gradually  and  at  sufficient 
intervals.  Horses  may  be  more  easily  immunized.  The  serum  of  such 
actively  immunized  animals  contains  agglutinins  in  considerable  con- 
centration and  of  a  specificity  sufficiently  illustrated  in  the  preceding 
section  dealing  with  the  identification  of  the  various  species.  For 
diagnostic  purposes  in  human  beings,  the  agglutination  reaction,  accord- 
ing to  the  technique  of  the  Widal  reaction  for  typhoid  fever,  has  been 
utilized  by  Kruse31  and  others.  According  to  most  observers,  normal 
human  serum  never  agglutinates  dysentery  bacilli  in  dilutions  greater 
than  one  in  twenty,  while  the  serum  of  dysentery  patients  will  often  be 
active  in  dilutions  as  high  as  one  in  fifty. 

Bactericidal  substances  have  been  demonstrated  in  the  serum  of 
immunized  animals  as  well  as  in  that  of  diseased  human  beings.  These 
have  been  determined,  in  vitro,  by  Shiga,32  and,  by  the  intraperitoneal 
technique  of  Pfeiffer,  by  Kruse.33  Bacteriolysis  may  take  place  in  high 
dilutions  of  the  serum,  and  has  recently  been  used  for  the  differentiation 
of  the  types  of  the  dysentery  bacilli  by  Ohno.34 

True  antitoxins  in  immune  sera  have  been  recently  described  by 
Kraus  and  Doerr.35 

Todd  has  demonstrated  that  the  mixture  of  such  an  immune  serum 
with  solutions  of  toxin  and  exposure  of  the  mixture  at  37.5°  C.  for  a 
half  hour  would  produce  almost  complete  neutralization  of  the  poison, 
thus  demonstrating  that  at  least  a  large  part  of  the  beneficial  action 
of  the  immune  sera  was  due  to  a  true  antitoxic  process.  Because  of 
the  different  varieties  of  dysentery  bacilli,  polyvalent  serum  has  been 
recommended. 

The  results  of  Olitsky  and  Kligler  are  even  better  than  this,  in  that 
they  have  succeeded  in  protecting  rabbits  against  1000  lethal  doses  of 
the  poison  with  their  antitoxic  horse  serum. 

31  Kruse,  Deut.  med.  Woch.,  1901. 

32  Shiga,  Zeit.  f.  Hyg.,  xli. 

33  Kruse,  Deut.  med.  Woch.,  1903. 

34  Ohno,  Philippine  Jour,  of  Sci.,  vol,  i,  1906. 
36  Kraus  und  Doerr,  loc.  cit. 


BACILLI    OF    THE    COLON-TYPHOID-DYSENTERY    GROUP     711 

DYSENTERY  IN  MAN 

The  clinical  term  " dysentery"  is  a  vague  one  and  may  signify 
violent  diarrheal  disturbances  from  almost  any  cause.  Technically, 
the  term  dysentery  should  be  restricted  either  to  the  arncebic  variety  or 
the  bacillary.  Bacterial  dysenteries  have  been  attributed  to  many 
different  organisms  besides  the  true  dysentery  bacilli,  such  as  some  of 
the  paratyphoid  bacilli,  B.  pyocyaneus,  the  Morgan  bacilli,  etc. 

Endemic  in  a  large  part  of  the  world,  especially  in  the  warmer 
climates,  the  disease  most  frequently  occurs  in  epidemics  of  more  or 
less  definite  localization,  usually  under  conditions  which  accompany 
the  massing  of  a  large  number  of  human  beings  in  one  place,  such  as 
those  which  occur  in  the  crowded  quarters  of  unsanitary  towns,  in  insti- 
tutions such  as  insane  asylums,  or  in  military  camps.  The  mortality  of 
such  epidemics  may  be  very  large.  According  to  Shiga,  the  disease  in 
Japan  frequently  shows  a  mortality  of  over  20  per  cent. 

The  disease  in  human  beings  usually  begins  as  an  acute  gastro- 
enteritis which  is  accompanied  by  abdominal  pain  and  diarrhea.  As 
it  becomes  more  severe,  the  colicky  pains  and  diarrhea  increase,  the 
stools  lose  their  fecal  character,  becoming  small  in  quantity  and  filled 
with  mucus  and  flakes  of  blood.  There  is  often  severe  tenesmus  at 
this  stage,  and  the  bacilli  are  present  in  large  numbers  in  the  dejecta. 
Owing  to  the  absorption  of  toxic  products,  symptoms  referable  to  the 
nervous  system,  such  as  muscular  twitching,  may  supervene,  and  if  the 
disease  is  at  all  prolonged,  there  are  marked  inanition  and  prostration. 

At  autopsy  in  early  stages  there  may  be  found  only  a  severe  catarrhal 
inflammation  of  the  mucous  membrane  of  the  large  intestine.  In 
the  later  stages  there  are  extensive  ulcerations,  and  the  bacteria  are 
found  lodged  within  the  depths  of  the  mucosa  and  submucosa.  Occa- 
sionally they  may  penetrate  to  the  mesenteric  glands,  but  as  far  as  we 
know  there  is  no  penetration  into  the  general  circulation. 

Although  this  acute  disease  represents  the  typical  picture  of  clinical 
dysentery,  it  must  not  be  forgotten  that  bacteria  of  the  dysentery 
types  may  cause  very  much  milder  intestinal  inflammations  and  even 
simple  diarrheas.  "Y"  bacilli  and  Flexner  bacilli  have  often  been 
isolated  from  such  mild  conditions,  especially  during  the  hot  weather. 

Epidemiology  and  Prevention. — Bacillary  dysentery  is  not  limited 
to  any  particular  part  of  the  world.  Unlike  the  ameobic  variety,  it  is 
probably  just  as  common  in  temperate  climates  as  it  is  in  the  tropical 
ones,  though  actual  epidemic  occurrence  is  probably  a  little  more  fre- 
quently observed  in  tropical  communities  where  fly  suppression  and 


712  PATHOGENIC   MICROORGANISMS 

sewage  and  garbage  disposal  have  not  been  developed  to  a  satisfactory 
extent. 

Dysentery  epidemics  also  are  more  apt  to  occur  during  the  hot  and 
dry  parts  of  the  year  when  flies  are  prevalent.  It  is  a  mistake  to  think 
of  the  disease  only  as  occurring  in  epidemics,  since  organisms  of  the 
dysentery  group  probabty  cause  a  great  many  sporadic  cases  and  small 
group  attacks  of  diarrheal  diseases  which  in  their  clinical  manifestations 
cannot  be  strictly  classified  as  dysentery."  Thus,  numerous  small  group 
outbreaks  have  been  studied  in  America  and  Europe,  occurring  either 
in  cities  sometimes,  as  in  those  studied  by  Kruse  and  others,  in  public 
institutions  as  insane  asylums  and  orphanages,  and  in  connection  with 
such  outbreaks  a  great  many  dysentery-like  organisms  have  been 
described.  At  times,  however,  the  disease  has  caused  widespread 
epidemic  outbreaks,  and  Castellani  and  Chalmers  36  mention  great 
epidemics  which  occurred  in  Europe  in  1538,  1777,  1779,  and  1834.  In 
its  epidemic  form  it  is  particularly  a  disease  of  armies.  Vincent  and 
Muratet 37  state  that  a  destructive  epidemic  took  place  in  the  English 
Armies  in  1415  after  the  battle  of  Agincourt.  There  were  serious  epi- 
demics in  the  armies  of  the  Allies  during  the  Crimean  War,  during  the 
American  Civil  War,  during  the  Franco-Prussian  War,  the  Russo-Turkish 
and  the  Russo-Japanese  War.  Considerable  dysentery  morbidity  occurred 
in  the  South  African  War,  and  during  the  recent  World  War  the  disease 
was  prevalent  among  all  the  armies  fighting  on  the  Eastern  and  Western 
fronts.  In  speaking  of  the  dysentery  morbidity  under  war  conditions, 
it  is  always  important  to  remember  the  almost  insuperable  difficulties 
which  render  accurate  diagnosis  of  the  disease  almost  impossible.  Also, 
it  is  likely  that  large  epidemics  are  rarely  caused  by  a  single  dysentery 
type. 

In  1905  Amako38  studied  the  epidemics  which  occurred  in  the  town 
of  Kobe  and  from  743  cases  isolated  dysentery  bacilli  in  526.  During 
this  single  epidemic  he  found  5  different  types  of  dysentery  bacilli, 
the  first  type  being  the  typical  Shiga  bacillus,  the  second  being  a  mannite 
fermenter,  the  fourth  and  fifth  fermenting  maltose  and  dextrin,  the 
third  having  no  effect  upon  maltose  and  dextrin  but  fermenting  sac- 
charose like  the  fourth  and  fifth.  He  found  the  first  type  in  108  cases, 
the  second  in  202,  the  third  type  in  9,  the  fourth  in  169,  and  the  fifth  in 

36  Castellani  and  Chalmers,  Text- book  of  Tropical  Medicine. 

37  Vincent  and  Muratet.  Dysentery,  etc.,  Military  Med.  Manual,  Univ.  of  London 
Press,  1917. 

w  Amako,  Zeit.  f.  Hyg.,  60,  1908,  93. 


BACILLI   OF   THE   COLON-TYPHOID-DYSENTERY    GROUP     713 

16  cases.  Such  investigations  amply  prove  that  even  local  epidemics 
may  be  caused  by  a  considerable  number  of  different  organisms.  In 
one  case,  as  a  matter  of  fact,  he  found  two  different  types  in  the  same 
patient.  Studying  family  epidemics  at  the  same  time,  he  usually  found 
one  and  the  same  type  in  a  single  family,  but  in  six  families  in  which 
there  were  25  patients  he  found  two  different  types. 

As  we  shall  see,  dysentery  is  transmitted  by  much  the  same  agencies 
which  are  responsible  for  transmission  of  other  intestinal  diseases, 
typhoid,  paratyphoid,  etc.,  and  in  consequence,  sanitary  and  other  con- 
ditions which  bring  about  one  disease  give  rise  to  cases  of  the  other. 
It  is  a  noticeable  feature  of  the  outbreaks  of  intestinal  disease  which 
occurred  in  the  Allied  Armies  during  the  late  war  that  every  outbreak 
of  dysenteric  maladies  was  accompanied  by  enormous  numbers  of  mild 
diarrheal  conditions.  This  is  mentioned  by  Vincent  and  Muratet  for 
the  French  and  British  Armies,  and  was  noticed  by  us  during  the  out- 
break of  similar  conditions  among  the  American  troops  in  July,  1918. 
At  such  times  the  intestinal  disturbances  are  almost  universal  among 
troops,  taking,  in  most,  the  form  of  a  mild  temporary  and  recurring 
diarrhea,  in  others  a  more  severe  diarrhea  with  fever,  and  in  others, 
again,  severe  symptoms  of  typical  dysentery.  Bacteriological  analysis 
of  large  numbers  of  cases  is  next  to  impossible  under  such  conditions, 
but  even  the  limited  number  made  during  the  late  war,  at  least  in  the 
zone  of  the  American  Armies  showed  that  all  the  known  varieties  of 
intestinal  invaders,  typhoid,  paratyphoid  and  the  various  dysentery 
bacilli,  played  a  role  in  the  outbreaks.  It  is  not  at  all  impossible  that 
many  of  the  mild  cases  may  have  been  true  dysentery  or  even  true 
typhoid,  modified  by  increased  natural  resistance  and  by  vaccination  in 
the  men.  On  the  other  hand,  it  is  also  quite  likely,  in  fact  is  the  view 
we  favor,  that  the  large  majority  of  the  mild  cases  which  constituted 
perhaps  90  per  cent  of  the  total,  represented  infections  by  various  other 
bacterial  agencies  originating  in  the  massive  infection  of  food  and  water 
with  fecal  organisms,  the  result  of  open  latrines,  limited  water  supplies 
and  active  fly  transmission. 

The  most  severe  epidemics  are  probably  those  due  to  the  Shiga 
bacillus.  However,  the  Flexner  bacilli  have  been  known  to  cause  con- 
siderable epidemics  in  southeastern  Europe  and  in  parts  of  Asia,  in  the 
Philippines,  Japan,  China  and  Ceylon.  The  Strong  bacillus  has 
been  known  to  cause  disease  in  the  Philippines  and  the  "Y"  bacillus 
has  been  found  rather  frequently  in  milder  outbreaks  and  in  sporadic 
cases  especially  in  the  United  States.  The  "  Y"  bacillus  has  been  par- 


714  PATHOGENIC   MICROORGANISMS 

ticularly  found  in  connection  with  cases  of  so-called  infantile  diarrhea, 
and  it  is  not  impossible  that  many  cases  of  diarrhea  in  children  may  be 
due  to  this  organism. 

In  many  countries  there  seem  to  be  endemic  foci  of  dysentery.  This 
has  been  particularly  studied  in  France  where  outbreaks  of  dysentery 
seem  often  to  have  started  in  the  central  part  in  the  neighborhood  of 
Tours,  and  it  is  not  unlikely  that  in  our  own  country  there  may  be  scat- 
tered dysentery  foci  in  the  large  cities  of  the  north  and  in  some  parts  of 
the  south. 

Dysentery  may  be  conveyed  by  a  number  of  different  agencies.  It 
is  easy  to  understand  that  in  a  disease  in  which  the  movements  become 
fluid  and  very  frequent,  and  in  which  many  mild  unhospitalized  cases 
may  exist,  the  scattering  of  infectious  material  is  very  much  more 
important  epidemiologically  than  it  would  be  in  a  disease  like  typhoid 
fever.  In  consequence,  transmission  from  man  to  man  by  hands  and 
indirect  contamination  of  food,  etc.,  is  common  during  epidemics.  The 
organisms  may  remain  alive  for  a  considerable  period  in  the  soil,  as  we 
have  seen,  and  under  camp  conditions,  infected  latrines  may  contam- 
inate water  supplies  and  by  the  intervention  of  flies,  scatter  the  organ- 
isms to  the  food.  Fly  transmission  is  of  the  utmost  importance  in 
dysentery  among  armies  in  the  hot  weather.  It  was  probably  the  most 
important  means  of  transmission  during  the  American  Army  epidemic 
spoken  of  above.  Large  water  epidemics,  as  described  in  the  case  of 
typhoid  fever,  are  uncommon,  although  isolated  ones  have  been 
described. 

Dysentery  carriers  unquestionably  exist.  The  organisms  may 
persist  for  months  in  the  intestinal  canals  of  convalescents  and  have 
been  described  in  the  stools  of  individuals  who  give  no  history  of  having 
had  the  disease.  The  carrier  problem  in  dysentery  is  a  very  difficult 
one  because  the  isolation  of  small  numbers  of  dysentery  bacilli  from 
stools  is  even  more  difficult  than  in  the  case  of  typhoid  bacilli.  It  has 
been  suggested  that  chronic  carriers  may  harbor  the  organisms  in  the 
gall  bladder,  but  this  is  not  definite. 

Shiga  who  has  made  extensive  epidemiological  studies  on  dysen- 
tery, expresses  the  opinion  that  the  chief  element  in  the  spread  of 
dysentery  is  the  carrier.  The  first  positive  carrier  examination 
was  made  by  Conradi39  who  isolated  dj^sentery  bacilli  from  the 
stools  of  three  perfectly  healthy  children  during  the  occurrence  of  a  con- 

39  Conradi,  Festschr.  fur  Robt.  Koch,  Jena,  1903,  cited  from  Lentz,  Kolle  and 
Wassermann,  2d  ed.,  vol.  3. 


BACILLI    OF    THE    COLON-TYPHOID-DYSENTERY    GROUP     715 

tact  epidemic  in  Metz.  Martha  Wollstein40  found  typical  dysentery 
bacilli  at  autopsy  in  the  intestines  of  children  who  had  presented  none 
of  the  symptoms  of  dysentery  before  death,  and  similar  isolations  were 
made  by  Duval  and  Shorer  41  in  connection  with  epidemics  of  summer 
diarrhea.  Shiga  claims  that  in  every  dysentery  epidemic  a  great  many 
contact  cases  can  be  traced  epidemiologically.  His  belief  is  that  it  is 
contact  which  keeps  the  disease  going  almost  entirely  during  the  inter- 
epidemic  periods  in  Japan,  and  that  this  is  largely  due  to  the  carrier 
condition  in  healthy  people,  mild  sporadic  cases  which  clinically  are 
diarrheal,  and  convalescents  of  typical  dysentery  cases.  Water,  he 
thinks,  can  play  a  part,  but  in  his  accounts  of  water  epidemics  this 
implies  rather  gross  carelessness  in  the  care  of  water.  It  was  sug- 
gested during  recent  epidemics  in  the  British  Army  that  dried  feces 
carried  about  by  dust  in  sand  storms  may  have  contributed  to  the 
spread  of  epidemics. 

Whether  or  not  domestic  animals  may  act  as  carriers  is  not  cer- 
tain, but  it  has  been  suggested  that  dogs  may  be  spontaneously  infected 
with  bacillary  dysentery  and  transmit  the  disease  to  human  beings. 

Castellani  mentions  that  Kruse  and  Bowman42  have  reported  spon- 
taneous bacillary  dysentery  in  monkeys  in  which  Flexner  bacilli  were 
isolated,  and  Messerschmidt43  found  "Y"  bacilli  in  the  feces  of 
healthy  rabbits.  Indirect  transmission  by  means  of  food  is  of  course, 
to  be  expected.  A  small  epidemic  occurring  in  a  hospital  in  New  York 
city  and  caused  by  the  bacillus  "Y, "  was  indirectly  traced  to  milk  by 
ourselves.44 

The  length  of  time  during  which  the  bacilli  may  live  in  the  soil 
has  been  mentioned  as  a  source  of  danger  by  a  number  of  writers, 
and  Vincent  and  Muratet  mention  a  case  which  seems  to  us  of 
considerable  importance  in  dealing  with  army  epidemics,  since  it- 
indicates  the  danger  of  bringing  new  troops  to  old  camping  grounds 
cither  in  the  course  of  advances  over  enemy  territory  or  in  the 
periodical  use  of  cantonments.  They  state  that  dysentery  had  been 
common  at  the  Chalons  Military  Camp  in  1889.  A  year  later,  troops 
coming  to  this  camp  pitched  their  tents  and  ditched  them  over  the 


"Wollsiein,  Martha,  Stud,  from  the  Rock.  Inst.,  2,  1904. 

41  Dtirnl  :md  Shorer ,  Stud,  from  the  Rock.  Inst.,  2,  1934. 

42  Kriise  and  Bowman,  cited  from  Castellani  and  Chalmers,  loc.  cit. 
4:<  Mesxerschmidt,  cited  from  Lentz,  loc:  cit. 

44  Zinsser,  Proc.  N.  Y.  Path.  Soc.,  1907, 


716  PATHOGENIC   MICROOKG  VNISMS 

site  of  the  old  latrines.  Dysentery  appeared  among  this  particular 
troop  unit,  whereas,  other  troops  remained  free. 

Preventive  measures  must  center  chiefly  upon  disinfection  of 
dejecta,  closure  of  latrines,  fly-proofing  of  latrines  and  kitchens,  early 
recognition  of  suspicious  cases  with  appropriate  measures  and 
enforced  cleanliness  of  food  handlers  and  of  all  men  before  and 
after  defecation.  Care  of  water  supplies  and  food  supplies  is  of 
course  indicated.  It  is  also  important  to  inquire  into  the  possibility 
of  recent  intestinal  disturbances  however  mild,  among  the  kitchen 
personnel  and  others  in  contact  with  food.  It  is  not  uncommon  in 
armies  to  find  that  company  cooks  are  suffering  from  mild  intestinal 
disturbances  to  which  they,  themselves,  have  paid  little  attention, 
but  which  may  be  sources  of  great  danger  to  others. 

Prophylactic  Vaccination. — Many  different  methods  of  prophy- 
lactic vaccination  have  been  attempted.  Early  attempts  were  dis- 
couraging because  of  the  violent  reactions  obtained  by  the  injection 
of  killed  broth  cultures  or  even  killed  suspensions  of  agar  growths. 

Shiga45  carried  out  vaccination  experiments  very  largely  because 
he  believed  that  the  periodical  appearance  of  epidemics  in  Japan- 
indicated  the  acquisition  of  immunity  by  the  community.  He  states, 
for  instance,  that  in  the  thirty  years  preceding  1908,  two  great 
epidemics  passed  through  Japan  from  the  south  to  the  north.  The 
epidemic  remained  in  a  single  region  from  one  to  three  years,  rapidly 
reaching  a  maximum  and  gradually  declining.  After  a  period  vary- 
ing from  ten  to  twenty  years,  these  great  epidemics  reappeared  and 
he  believed  that  the  interval  could  be  explained  by  acquired  im- 
munity. Shiga's  method  consisted  very  largely  in  injecting,  simul- 
taneously, killed  suspensions  of  agar  cultures  with  specific  serum  (or 
in  other  words,  sensitized  cultures),  following  this  first  injection  with 
killed  agar  suspensions  without  serum.  This  had  the  advantage, 
which  sensitized  cultures  always  have,  of  not  giving  the  severe 
reactions  which  follow  the  injections  of  cultures  alone.  By  these 
methods  he  vaccinated  10,000  Japanese  in  epidemic  regions  without 
very  striking  results  as  far  as  the  morbidity  is  concerned,  but  with 
considerable  lowering  of  the  mortality. 

Castellani  inoculated  0.5  c.c.  of  a  peptone  water  culture,  killed 
by  heating  to  55°  for  one  hour,  following  this,  after  a  week,  with  a 
second  inoculation  of  1  c.c.  Most  observers  have  worked  with  killed 

45  Shiga,  Cent,  f .  Bakt.,  Orig.,  42,  1909,  132. 


BACILLI    OF    THE    COLON-TYPHOID-DYSENTERY    GROUP      717 

suspensions  of  agar  cultures  taken  up  in  salt  solution.  The  statistics 
of  vaccinations  done  during  the  late  war  in  isolated  troop  unit  are  not 
available  at  the  present  writing.  Systematic  dysentery  vaccination 
was  not,  however,  carried  out  in  any  of  the  European  armies  as  far  as 
we  know.  The  entire  subject  is  still  in  its  experimental  stages  and  no 
conclusive  statements  can  be  made  regarding  it.  As  a  matter  of  fact, 
judging  from  the  immunological  conditions  prevailing  in  the  disease 
and  the  localization  of  the  organisms,  one  would  expect  just  what 
Shiga  found,  a  reduction  of  the  mortality  without  any  considerable 
diminution  of  the  morbidity. 

Serum  Treatment. — Sera,  both  monovalent  and  polyvalent,  have 
been  made  by  a  large  number  of  observers  and  extensive  attempts 
at  treatment  have  been  carried  out.  Shiga  himself  used  a  mul- 
tivalent  dysentery  serum  in  which  he  used  the  various  types  isolated 
in  Japan.  He  obtained  very  encouraging  results  in  thousands  of 
cases  in  Japan,  and  believed  that  there  was  a  very  definite  thera- 
peutic advantage  to  be  gained  by  use  of  the  serum.  Other  reports 
have  been  conflicting.  If  dysentery  serum  is  to  be  of  great  value, 
it  probably  will  be  most  valuable  in  the  Shiga  types  of  the  disease 
in  which  an  exotoxin  seems  definitely  to  occur.  Sera  like  those 
produced  by  Olitsky  and  Kligler  have  not  yet  been  used  with  suffi- 
cient extensiveness  to  warrant  final  judgment. 

THE  MORGAN  BACILLI 

In  1905  and  1906  Morgan46  made  a  systematic  study  of  the 
bacteriology  of  diarrheal  diseases  in  infancy.  He  made  a  large 
number  of  isolations  from  stools  of  such  children,  and,  among  other 
things,  isolated  from  a  number  of  cases  types  of  Gram-negative 
bacilli,  obviously  belonging  into  the  paratyphoid-dysentery  group 
which,  however,  did  not  correspond  exactly  with  species  previously 
described. 

Morgan's  bacillus  I,  he  isolated  from  twenty-eight  cases  of  infant 
diarrhea,  out  of  fifty-eight  examinations,  and  in  seventeen  of  these 
it  was  the  only  lactose  non-fermenting  organism  present.  This 
organism,  was  motile,  produced  acid  and  gas  on  glucose,  but  did 
not  ferment  mannite,  dulcite,  lactose  and  saccharose.  It  differed 
from  the  hog  cholera  bacillus  in  producing  an  alkalin  reaction  on 

46  Morgan,  H.  de  #.,  Brit.  Med.  Jour.,  1,  1906,  908. 


718 


PATHOGENIC   MICROORGANISMS 


litmus  milk  and  in  giving  a  powerful  indol  reaction.  It  produced 
neither  acid  nor  gas  on  maltose  and  dextrin.  It  differed  from  most 
other  indol  producing  organisms  in  that  it  produced  this  substance 
in  glucose  broth  in  the  presence  of  excessive  glucose,  and  by  dis- 
tillation Morgan  proved  that  it  produced  indol  and  not  skatol- 
carboxylic  acid.  Morgan's  I  bacillus  is  the  only  one  of  his  bacilli 
in  which  pathogenicity  can  be  considered  as  a  possibility.  He  does 
not  draw  any  definite  conclusions  in  this  regard.  He  is  inclined, 
however,  to  believe  that  the  No.  I  bacillus  may  have  pathogenic 
properties. 


COMPOSITE  TABLE  OF  MOST  IMPORTANT  TYPES 


Dex- 
trose. 

Man- 
nit. 

Mal- 
tose. 

Lac- 
tose. 

Xy- 
lose. 

Rham- 
nose. 

Sac- 
cha- 
rose 

Dul- 
cit. 

Lead 
ace- 
tate 
agar. 

Indol. 

Mo- 

tility. 

B.  coli  com'is  .  .  . 

© 

© 

© 

© 

X 

X 

_ 

© 

varies 

+ 

+ 

B.  coli  com'ior  .  . 

© 

© 

© 

© 

X 

X 

© 

© 

varies 

4- 

B.  acidi  lactici  .  . 

© 

© 

© 

© 

X 

X 

_ 

— 

4- 

B.  lactis  aerog.  . 

© 

© 

© 

© 

X 

X 

© 

— 

4- 

B.  parat.  "A".  . 

© 

© 

© 

— 

— 

4- 

— 

slow 

— 

O  j3  -*^   C 

C  60  O  £ 

4- 

B.  parat.  "B".  . 

© 

© 

_ 

-J. 

4- 

— 

4- 

4- 

Js«* 

4- 

B.  enteritidis.  .  . 

© 

© 

_ 

4_ 

4_ 

_ 

4. 

4. 

4_ 

B.  abortus  

© 

© 



_j_ 

_l_ 



_l_ 



'a  _  -g  .  to  ft 

_l_ 

B.  hog  cholera  .  . 

© 

© 

. 

— 

4- 

+ 

— 

irreg. 

— 

IJJfl 

4- 

B.  typhi  murium 

© 

© 

— 

4- 

4- 

— 

4- 

4- 

4- 

B.  typhosus.  .  .  . 

4- 

4- 

4- 

— 

slow 

— 

— 

slow 

4- 

— 

4- 

B.  dys.  Shiga.  .  . 

4- 

— 

— 

- 

X 

X 

— 

.— 

X 

— 

— 

B.  dys.    Flexner. 

4- 

4- 

4- 

— 

X 

X 

— 

— 

?X 

4- 

— 

B.      dys.       "Y" 

(Hiss-Park)... 

4- 

4- 

— 

— 

X 

X 

— 

X 

X 

4- 

— 

B.  dys.  Strong..  . 

•f 

4- 

— 

— 

X 

X 

4- 

X 

X 

+ 

— 

B.  faec.alkal.  .  .  . 

— 

X 

X 

X 

X 

X 

X 

X 

X 

4- 

B.  Morgan,  No.  1 

©  + 

- 

- 

- 

? 

? 

- 

- 

?x 

4- 

4- 

slight 

gas 

©  =  acid  and  gas. 
+  =acid,  no  gas. 


—  =  negative. 

X  =  not  needed  for  identification. 


Other  organisms  which  he  regards  as  possibly  important  he 
speaks  of  as  his  Nos.  Ill  and  IV  types,  both  of  which  resembled 
somewhat  the  Flexner  type  of  dysentery.  No.  Ill  he  says  agglu- 
tinated, equally  well  with  either  Flexner  or  typhoid  serum  as  if 
closely  related  to  both.  It  differed  from  the  Flexner  bacillus  in 
producing  acid  on  sorbite. 

His  No.  IV  differs  from  the  Flexner  in  not  producing  indol.    The 


BACILLI   OF   THE   COLON-TYPHOID-DYSENTERY   GROUP     719 

No.  IV  agglutinated  in  the  Flcxner  serum,  but  not  at  all  with  typhoid 
serum. 

It  would  be  confusing  to  go  more  deeply  into  the  Morgan  group. 
It  is  important,  however,  to  mention  them  since  his  work  illustrates 
the  possibility  of  there  being  a  large  number  of  insufficiently  differ- 
entiated types  of  Gram-negative  bacilli  in  the  intestines  of  human 
beings.  In  view  of  the  many  researches  of  recent  years  in  which 
the  possibility  of  variants  of  unknown  species  in  regard  to  sugar 
fermentations,  agglutlnability,  etc.,  has  been  pointed  out,  it  would 
seem  proper  to  advise  great  caution  in  accepting  as  new  species, 
organisms  which  differ  from  well-known  types  in  only  one  or  another 
characteristic. 


CHAPTER   XXXV 

BACILLUS  MUCOSUS  CAPSULATUS,  BHINOSCLEROMA  AND  OZ^ENA 
BACILLUS  MUCOSUS  CAPSULATUS 

(Bacterium  pneumonice,  Friedlander's  bacillus,  Pneumobacillus) 

IN  1882  Friedlander1  announced  the  discovery  of  a  micro- 
organism which  he  believed  to  be  the  incitant  of  lobar  pneumonia 
and  which,  in  his  original  communications,  he  described  as  a  "  micro- 
coccus/' 

A  superficial  morphological  resemblance  between  Friedlander's 
microorganism  and  Diplococcus  lanceolatus,  now  recognized  as  the 
most  frequent  cause  of  lobar  pneumonia,  led,  at  first,  to  much  confu- 
sion, and  it  was  not  until  several  years  later,  owing  to  the  careful 
researches  of  Frankel2  and  of  Weichselbaum,3  that  the  "micro- 
coccus"  of  Friedlander  was  recognized  as  a  short,  encapsulated 
bacillus  which  occurred  in  lobar  pneumonia  exceptionally  only. 
Similar  bacilli  were  subsequently  found  by  other  observers,  bacilli 
which,  upon  morphological  grounds,  are  classified  together  as  the 
"Friedlander  group,"  or  the  "group  of  Bacillus  mucosus  capsulatus." 

Morphology  and  Staining. — The  Friedlander  bacillus  is  a  short, 
plump  bacillus  with  rounded  ends,  subject  to  great  individual  varia- 
tions as  to  size.  Its  average  measurements  are  from  0.5  to  1.5  micra 
in  width  and  0.6  to  5  micra  in  length.  Forms  approaching  both 
extremes  may  be  met  with  in  one  and  the  same  culture.  The  short, 
thick  forms,  frequently  found  in  animal  and  human  lesions,  are 
almost  coccoid  and  account  for  Friedlander's  error  in  first  describing 
the  bacillus  as  a  micrococcus.  The  bacilli  may  be  single,  in  diplo- 
form,  or  in  short  chains.  They  are  non-motile  and  possess  no  flagella. 
Spores  are  not  formed. 

1  Friedlander,  Virohow's  Arch.,  Ixxxvii,  1882;    Fort.  d.  Med.,  i,  1883;    ibid.,  ii. 
1884. 

2  Frankel,  Zeit.  f.  klin.  Med.,  x,  1886. 

3  Weichselbaum,  Med.  Jahrb.,  Wien,  1886. 

720 


BACILLUS   MUCOSUS  CAPSULATUS  721 

The  bacillus  is  characteristically  surrounded  by  a  well-developed 
capsule  which  is  most  perfectly  demonstrated  in  preparations  taken 
directly  from  some  animal  fluid,  such  as  the  secretion  or  exudate 
from  infected  areas.  It  is  also  seen,  however,  in  smears  made  from 
agar  or  gelatin  cultures.  The  capsule  is  usually  large,  twice  or  three 
times  the  size  of  the  bacillus  itself.  When  seen  in  chains  or  in 
groups,  several  bacilli  may  appear  to  be  inclosed  in  one  capsule. 
Prolonged  cultivation  on  agar  or  gelatin  may  result  in  disappearance 
of  the  capsule,  The  bacillus  is  easily  stained  with  the  ordinary 


•     •      ' 

.    •    *  ' 

'•   •*  *:  v,      -  %, 
*..'.   -    %     xt. 

"^fc  W  ^  m 


»     . 

****.'  V»      « 


»   -    > 


FIG.  71. — BACILLUS  MUCOSUS  CAPSULATUS. 

dyes,  but  is  decolorized  when  stained  by  the  Gram-method.  Capsules 
may  often  be  seen  when  the  more  intense  anilin  dyes  are  employed. 
They  are  brought  out  with  much  regularity  by  any  of  the  usual 
capsule  stains. 

Cultivation. — B.  mucosus  capsulatus  is  easily  cultivated.  It  grows 
readily  on  all  the  usual  culture  media,  both  on  those  having  a  meat- 
infusion  basis  and  on  those  made  with  meat  extract.  Growth  takes 
place  at  room  temperature  (18°  to  20°)  and  more  rapidly  at  37.5°  C. 
A  temperature  of  60°  C.  and  over  kills  the  bacilli  in  a  short  time. 
The  thermal  death-point-  according  to  Sternberg  is  56°  C.  Growth 


722  PATHOGENIC   MICROORGANISMS 

ceases  below  10°  to  12°  C.  Kept  at  room  temperature  and  protected 
from  drying,  the  bacillus  may  remain  alive,  in  cultures,  for  several 
months. 

The  bacillus  is  not  very  fastidious  as  to  reaction  of  media,  grow- 
ing equally  well  on  moderately  alkaline  or  acid  media.  It  is  aerobic 
and  facultatively  anaerobic;  growth  under  anaerobic  conditions, 
however,  is  not  luxuriant. 

On  agar,  growth  appears  in  the  form  of  grayish-white  mucus-like 
colonies,  having  a  characteristically  slimy  and  semi-fluid  appearance. 
Colonies  have  a  tendency  to  confluence,  so  that  on  plates,  after  three 
or  four  days,  a  large  part  of  the  surface  appears  as  if  covered  with 
a  film  of  glistening,  sticky  exudate,  which,  if  fished,  comes  off  in  a 
tenacious,  stringy  manner.  It  is  often  possible  to  make  a  tentative 
diagnosis  of  the  bacillus  from  the  appearance  of  this  growth. 

In  broth,  there  is  rapid  and  abundant  growth,  with  the  formation 
of  a  pellicle,  general  clouding,  and  later  the  development  of  a  pro- 
fuse, stringy  sediment. 

Stab  cultures  in  gelatin  show,  at  first,  a  white,  thin  line  of  growth 
along  the  course  of  the  puncture.  Soon,  however,  rapid  growth  at 
the  top  results  in  the  formation  of  a  grayish  mucoid  droplet  on  the 
surface,  which,  enlarging,  gives  the  growth  a  nail-like  appearance. 
This  nail-shape  was  originally  described  by  Friedlander  and 
regarded  as  diagnostic  for  the  bacillus.  The  gelatin  is  not  fluidified. 
As  the  culture  grows  older  the  entire  surface  of  the  gelatin  tube 
may  be  covered  with  growth,  flowing  out  from  the  edges  of  the 
nail-head.  The  gelatin  acquires  a  darker  color  and  there  may  be 
a  few  gas  bubbles  below  the  surface.  Microscopically,  colonies  on 
gelatin  plates  have  a  smooth  outline  and  a  finely  granular  or  even 
homogeneous  consistency. 

On  blood  serum,  a  confluent  mucus-like  growth  appears. 

On  potato,  abundant  growth  appears,  slightly  more  brownish  in 
color  than  that  on  other  media. 

In  pepton  solutions,  there  is  no  indol  formation. 

In  milk,  there  is  abundant  growth  and  marked  capsule  develop- 
ment. Coagulation  occurs  irregularly. 

In  considering  the  general  cultural  characteristics  of  the  Fried- 
lander  bacillus,  it  must  not  be  forgotten  that  we  are  dealing  with  a 
rather  heterogeneous  group,  the  individuals  of  which  are  subject  to 
many  minor  variations.  Capsule  development,  lack  of  motility,  in- 
ability to  fluidify  gelatin,  failure  to  form  indol,  and  absence  of 


BACILLUS   MUCOSUS  CAPSULATUS  723 

spores,  are  characteristics  common  to  all.  In  size,  general  appear- 
ance, gas  formation,  and  pathogenicity,  individual  strains  may  vary 
much,  one  from  the  other.  Strong4  has  studied  various  races  as 
to  gas  formation  and  concludes  that  most  strains  form  gas  from 
dextrose  and  levulose,  but  that  lactose  is  fermented  by  some  only. 
About  two-thirds  of  the  gas  formed  is  hydrogen,  the  rest  C02.  Acid 
formation,  according  to  Strong,  is  also  subject  to  much  variation 
among  different  races.  Similar  studies  by  Perkins5  show  that  most 
of  the  ordinary  cultural  characteristics  of  bacilli  of  this  group  are 
extremely  variable  and  can  not  serve  as  a  basis  for  differentiation. 
Reactions  on  sugars,  however,  are  more  constant.  Perkins  suggests 
the  following  tentative  division  classes  on  this  basis: 

I.  All  carbohydrates  fermented  with  the  formation  of  gas. 

II.  All  carbohydrates,  except  lactose,  fermented  with  the  forma- 
tion of  gas. 

III.  All  carbohydrates,   except  saccharose,   fermented  with  the 
formation  of  gas. 

Type  I.  corresponds  to  B.  aerogenes  (Migula),  type  II.  to  B. 
Friedlander  or  Bacterium  pneumoniae  (Migula),  and  type  III.  to 
Bacillus  lactis  aerogenes. 

Differentiation  by  means  of  serum  reactions  has  not  proved  satis- 
factory.6 

Pathogenicity. — When  Friedlander  first  described  this  micro- 
organism, he  assumed  it  to  be  the  incitant  of  lobar  pneumonia.  Sub- 
sequent researches  by  Weichselbaum7  and  others  have  shown  it  to 
be  etiologically  associated  with  pneumonia  in  about  seven  or  eight 
per  cent  of  all  cases.  The  percentage  in  this  country  is  probably 
lower.  Such  cases  can  often  be  diagnosed  by  the  presence  of  the 
bacilli  in  the  sputum,  which  is  peculiarly  sticky  and  stringy.  Cases 

4  Strong,  Cent,  f .  Bakt.,  xxv,  1899. 

5  Perkins,  Jour.  of.  Infect.  Dis.,  I,  No.  2,  1904. 

6  J.  G.  Fitzgerald,  who  has  recently  made  a  careful  study  of  the  mucosus  cap- 
sulatus  group  has  concluded  that  present  methods  do  not  permit  a  subdivision  of 
these  organisms  into  separate  species.     He  offers  the  following  "tentative  sug- 
gestion": "It  is  conceivable  that  mutations  based  on  the  necessity  of  maintaining  a 
parasitic  existence  have  caused  Gram-negative  bacilli  found  normally  in  the  body 
elsewhere  than  in  the  intestinal  tract  to  develop  capsules  for  protection  and  a  new 
group  has  arisen  which  we  designate  B.  mucosus  capsulatus;    and  the  varieties  B. 
aerogenes  and  B.  acidi  lactici  connect  the  group  with  the  non-encapsulated  colon 
group." 

7  Weichselbaum,  loc.  cit. 


724  PATHOGENIC   MICROORGANISMS 

of  Friedlander  pneumonia  are  extremely  severe  and  usually  fatal. 
The  bacillus  has  been  found  in  cases  of  ulcerative  stomatitis  and 
nasal  catarrh;  in  two  cases  of  severe  tonsillitis  in  children;  in  the 
pus  from  suppurations  in  the  antrum  of  Highmore  and  the  nasal 
sinuses  (Frankel  and  others),  and  in  cases  of  fetid  coryza  (ozena), 
of  which  disease  it  is  supposed  by  Abel8  and  others  to  be  the  specific 
cause.  Whether  the  ozena  bacillus  represents  a  separate  species 
or  not,  can  not  at  present  be  decided.  The  bacillus  of  Friedlander 
has  been  found  in  empyema  fluid,  in  pericardial  exudate  (after 
pneumonia),  and  in  spinal  fluid.9  Isolated  cases  of  Friedlander 
bacillus  septicemia  have  been  described.10  Being  occasionally  a 
saprophytic  inhabitant  of  the  normal  intestine,  it  has  been  believed 
to  be  etiologically  associated  with  some  forms  of  diarrheal  enteritis. 

B.  mucosus  capsulatus  is  pathogenic  for  mice  and  guinea-pigs, 
less  so  for  rabbits.  Inoculation  of  susceptible  animals  is  followed 
by  local  inflammation  and  death  by  septicemia.  If  inoculation  is 
intraperitoneal,  there  is  formed  a  characteristically  mucoid,  stringy 
exudate. 

The  question  of  immunization  against  bacilli  of  the  Friedlander 
group  is  still  in  the  stage  of  experimentation.  Immunization  with 
carefully  graded  doses  of  dead  bacilli  has  been  successful  in  isolated 
cases.  Specific  agglutinins  in  immune  serum  have  been  found  by 
Clairmont,11  but  irregularly  and  potent  only  against  the  particular 
strain  used  for  the  immunization. 

OTHER  BACILLI  OF  THE  FRIEDLANDER  GROUP 

Bacillus  of  Rhinoscleroma.— This  bacillus,  described  by  v. 
Frisch12  in  1882,  is  a  plump,  short  rod,  with  rounded  ends,  mor- 
phologically almost  identical  with  Friedlander 's  bacillus;  it  is  non- 
motile  and  possesses  a  distinct  capsule.  Although  at  first  described 
as  Gram-positive,  it  has  been  shown  to  be  decolorized  with  this 
method  of  staining.  It  forms  slimy  colonies,  has  a  nail-like  appearance 
in  gelatin  stab  cultures,  and  in  pepton  solutions  produces  no  indol.  It 
differs  from  B.  mucosus  capsulatus  (Wilde13)  in  forming  no  gas  in 

M6eZ,  Zeit.  f.  Hyg.,  xxi. 

9  Jager,  Zeit.  f .  Hyg.,  xix. 

10  Howard,  Johns  Hopkins  Hosp.  Bull.,  1899. 

11  Clairmont,  Zeit.  f.  Hyg.,  xxxix. 

12  Frisch,  Wien.  med.  Woch.,  1882. 

13  Wilde,  Cent,  f .  Bakt.,  xx,  1896. 


OTHER  BACILLI  OF  THE  FRIEDLANDER  GROUP  725 

dextrose  bouillon,  in  producing  no  acid  in  lactose  bouillon,  and  in 
never  coagulating  milk. 

Pathogenicity. — The  bacillus  of  rhinoscleroma  is  but  moderately 
pathogenic  for  animals  delicately  susceptible  to  the  bacillus  of  Fried- 
lander.  Rhinoscleroma,  the  disease  produced  by  this  bacillus  in  man, 
consists  of  a  slowly  growing  granulomatous  inflammation,  located 
usually  at  the  external  nares  or  upon  the  mucosa  of  the  nose,  mouth, 
pharynx,  or  larynx.  It  is  composed  of  a  number  of  chronic,  hard, 


FIG.  72. — BACILLUS  OF  RHINOSCLEROMA.     Section  of  tissue  showing  the  micro- 
organisms within  Mikulicz  cells.     (After  Frankel  and  Pfeiffer.) 

nodular  swellings,  which,  on  histological  examination,  show  granu- 
lation tissue  and  productive  inflammation.  In  the  meshes  of  the 
abundant  connective  tissue  lie  many  large  swollen  cells,  the  so-called 
' '  Mikulicz  cells. ' ' 14  The  rhinoscleroma  bacilli  lie  within  these  cells 
and  in  the  intercellular  spaces.  They  can  be  demonstrated  in  his- 
tological sections  and  can  be  cultivated  from  the  lesions,  usually 
in  pure  culture.  Rhinoscleroma  is  rare  in  America.  It  is  most 
prevalent  in  Southeastern  Europe.  The  disease  is  slowly  progressive 
and  comparatively  intractable  to  surgical  treatment,  but  hardly  ever 
affects  the  general  health  unless  by  mechanical  obstruction  of  the 
air  passages. 

14  Mikulicz,  Arch.  f.  Chir.,  xx,  1876. 


726  PATHOGENIC   MICROORGANISMS 

B.  Ozaense. — The  work  of  Abel15  and  others  has  shown  that  ozena, 
or  fetid  nasal  catarrh,  is  almost  always  associated  witli  a  bacillus 
morphologically  and  culturally  almost  identical  with  B.  mucosus 
capsulatus.  The  bacillus  can  not  be  definitely  separated  from  the 
latter.  According  to  Wilde  it  forms  no  gas  in  dextrose  bouillon 
and  is  less  pathogenic  for  mice  than  B.  Friedlander.  Whether  it  is 
a  separate  species,  or  merely  an  atypical  form  changed  by  environ- 
ment, can  not  be  stated  at  present. 

Perez  Bacillus  of  Ozsena. — Perez16  in  1899  described  another 
microorganism  which  he  connects  etiologically  with  ozaena.  The 
Perez  bacillus  is  Gram-negative,  pleomorphic,  non-motile  and  non- 
capsulated.  It  grows  easily  on  ordinary  media,  does  not  liquefy 
gelatin,  and  makes  indol.  Its  cultures  have  a  characteristic  fetid 
odor.  Intravenously  injected  into  rabbits  it  seems  to  produce  a 
localized  lesion  in  the  nasal  cavity  on  the  turbinated  bones.  Hofer17 
has  also  isolated  it,  but  recent  work  leaves  its  importance  as  the 
causative  agent  in  doubt. 

16  Abel,  Zeit.  f.  Hyg.,  xxi. 

18  Perez,  Animal  de  PInst.  Past.  1899. 

17  Hofer,  Wien.  klin.  Woch.,  vol.  26,  pp.  1011  and  1628. 


CHAPTER   XXXVI 

THE     ANAEROBIC     BACILLI.     TETANUS     AND     BACILLUS  TETANI. 
BOTULISMUS  AND  THE  BACILLUS  BOTULINUS 

LOCKJAW  or  tetanus, 'though  a  comparatively  infrequent  disease, 
has  been  recognized  as  a  distinct  clinical  entity  for  many  centuries. 
The  infectious  nature  of  the  disease,  however,  was  not  demonstrated 
until  1884,  when  Carlo1  and  Rattone  succeeded  in  producing  tetanus 
in  rabbits  by  the  inoculation  of  pus  from  the  cutaneous  lesion  of 
a  human  case.  Nicolaier,2  not  long  after,  succeeded  in  producing 
tetanic  symptoms  in  mice  and  rabbits  by  inoculating  them  with  soil. 
In  connection  with  the  lesions  produced  at  the  point  of  inoculation, 
Nicolaier  described  a  bacillus  which  may  have  been  Bacillus  tetani, 
but  which  he  was  unable  to  cultivate  in  pure  culture.  Kitasato,3 
in  1889,  definitely  solved  the  etiological  problem  by  obtaining  from 
cases  of  tetanus  pure  cultures  of  bacilli  with  which  he  was  able 
again  to  produce  the  disease  in  animals. 

Kitasato  succeeded  where  others  had  failed  because  of  his  use  of 
anaerobic  methods  and  his  elimination  of  non-spore-bearing  con- 
taminating organisms  by  means  of  heat.  His  method  of  isolation 
was  as  follows :  The  material  containing  tetanus  bacilli  was  smeared 
upon  the  surface  of  agar  slants.  These  were  permitted  to  develop 
at  incubator  temperature  for  twenty-four  to  forty-eight  hours.  At 
the  end  of  this  time  the  cultures  were  subjected  to  a  temperature 
of  80°  C.  for  one  hour.  The  purpose  of  this  was  to  destroy  all 
non-sporulating  bacteria,  as  well  as  aerobic  spore-bearers  which  had 
developed  into  the  vegetative  form.  Agar  plates  were  then  in- 
oculated from  the  slants  and  exposed  to  an  atmosphere  from  which 
oxygen  had  been  completely  eliminated  and  hydrogen  substituted. 
On  these  plates  colonies  of  tetanus  bacilli  developed. 

Morphology  and  Staining. — The  bacillus  of  tetanus  is  a  slender 
bacillus,  2  to  5  micra  in  length,  and  0.3  to  0.8  in  breadth.  The 

1  Carlo  e  Rattone,  Giornale  d.  R.  Acad.  d.  Torino,  1884. 

2  Nicolaier,  Inaug.  Diss.,  Gottingen,  1885. 

8  Kitasato,  Deut.  med.  Woch.,  No.  xxxi,  1889. 

727 


728 


PATHOGENIC  MICROORGANISMS 


vegetative  forms  which,  occur  chiefly  in  young  cultures  are  slightly 
motile  and  are  seen  to  possess4  numerous  peritrichal  flagella,  when 
stained  by  special  methods.  After  twenty-four  to  forty-eight  hours 
of  incubation,  the  length  of  time  depending  somewhat  on  the  nature 
of  the  medium  and  the  degree  of  anaerobiosis,  the  bacilli  develop 
spores  which  are  characteristically  located  at  one  end,  giving  the 
bacterium  the  diagnostic  drumstick  appearance. 


FIG.  73. — BACILLUS  TETANI.     Spore  stain. 

As  the  cultures  grow  older  the  spore-bearing  forms  completely 
supersede  the  vegetative  ones.  Very  old  cultures  contain  spore- 
bearing  bacilli  and  spores  only. 

The  tetanus  bacillus  is  easily  stained  by  the  usual  amlin  dyes, 
and  reacts  positively  to  Gram's  stain.  Flagella  staining  is  successful 
only  when  very  young  cultures  are  employed. 

Distribution. — In  nature,  the  tetanus  bacillus  has  been  found  by 
Nicolaier  and  others  to  occur  in  the  superficial  layers  of  the  soil. 
The  earth  of  cultivated  and  manured  fields  seems  to  harbor  this 

4  Vottaler,  Zeit.  f .  Hyg.,  xxvii. 


THE  ANAEROBIC  BACILLI  729 

organism  with  especial  frequency,  probably  because  of  its  presence 
in  the  dejecta  of  some  of  the  domestic  animals. 

Biological  Characteristics. — The  bacillus  of  tetanus  is  generally 
described  as  an  obligatory  anaerobe.  While  it  is  unquestionably 
true  that  growth  is  ordinarily  obtained  only  in  the  complete  absence 
of  oxygen,  various  observers,  notably  Ferran5  and  Belfanti,6  have 
successfully  habituated  the  bacillus  to  aerobic  conditions  by  the 
gradual  increase  of  oxygen  in  cultures.  Habituation  to  aerobic  con- 
ditions has  usually  been  accompanied  by  diminution  or  loss  of 
pathogenicity  and  toxin-formation.  Anaerobic  conditions  may  like- 
wise be  dispensed  with  if  tetanus  bacilli  be  grown  in  symbiosis  with 
some  of  the  aerobic  bacteria.  The  addition  to  culture  media  of  suit- 
able carbohydrates,  and  of  fresh  sterile  tissue,  has  also  been  found  to 
render  it  less  exacting  as  to  mechanical  anaerobiosis.7 

Anaerobically  cultivated,  Bacillus  tetani  grows  readily  upon  meat- 
infusion  broth,  which  it  clouds  within  twenty-four  to  thirty-six  hours. 

Upon  meat-infusion  gelatin  at  20°  to  22°  C.  the  tetanus  bacillus 
grows  readily,  growth  becoming  visible  during  the  second  or  third 
day.  There  is  slow  liquefaction  of  the  gelatin. 

On  agar,  at  37.5°  C.,  growth  appears  within  forty-eight  hours. 
Colonies  on  agar.  plates  present  a  rather  characteristic  appearance, 
consisting  of  a  compact]  center  surrounded  by  a  loose  mesh  work  of 
fine  filaments,  not  unlike  the  medusa-head  appearance  of  subtilis 
colonies.  In  agar  stabs,  fine  radiating  processes  growing  out  in  all 
directions  from  the  -central  stab  tend  to  give  the  culture  the  appear- 
ance of  a  fluff  of  cotton.  Milk  is  a  favorable  culture  medium  and  is 
not  coagulated.  On  potato,  growth  is  delicate  and  hardly  visible. 

The  most  favorable  temperature  for  the  growth  of  this  bacillus 
is  37.5°  C.  Slight  alkalinity  or  neutrality  of  the  culture  media  is 
most  advantageous,  though  moderate  acidity  does  not  altogether 
inhibit  growth.  All  the  media  named  may  be  rendered  more  favor- 
able still  by  the  addition  of  one  or  two  per  cent  of  glucose,  maltose, 
or  sodium  formate.8  In  media  containing  certain  carbohydrates, 
tetanus  bacilli  produce  acid.  In  gelatin  and  agar,  moderate  amounts 
of  gas  are  produced,  consisting  chiefly  of  C02,9  but  with  the  admix- 

5  Ferran,  Cent.  f.  Bakt.,  xxiv,  No.  1. 

6  Belfanti,  Arch,  per  le  sci.  med.,  xvi. 

7  Th.  Smith,  Brown,  and  Walker,  Jour.  Med.  Res.,  N.  S.,  ix,  1906. 

8  Kitasato,  Ztschr.  f.  Hyg.  1891. 

9  v.  Eisler  and  Pribram  in  Kraus  and  Levaditi,  Handbuch,  etc.,  Jena,  1907, 


730 


PATHOGENIC   MICROORGANISMS 


tures  of  other  volatile  substances  which  give  rise  to  a  characteristic- 
ally unpleasant  odor,  not  unlike  that  of  putrefying  organic  matter. 
This  odor  is  due  largely  to  H2S  and  methylmercaptan. 

The  vegetative  forms  of  the  tetanus  bacillus  are  not  more  re- 
sistant against  heat  or  chemical  agents  than  the  vegetative  forms 
of  other  microorganisms,  Tetanus  spores,  however,  will  resist  dry 


FIG.  74. — YOUNG  TET- 
ANUS CULTURE  IN  GLU- 
COSE AGAR, 


FIG.  75. — OLDER  TET- 
ANUS CULTURE  IN  GLU- 
COSE AGAR. 


heat  at  80°  C.  for  about  one  hour,  live  steam  for  about  five  minutes; 
five  per  cent  carbolic  acid  kills  them  in  twelve  to  fifteen  hours; 
one  per  cent  of  bichlorid  of  mercury  in  two  or  three  hours.  Direct 
sunlight  diminishes  their  virulence  and  eventually  destroys  them. 
Protected  from  sunlight  and  other  deleterious  influences,  tetanus 
spores  may  remain  viable  and  virulent  for  many  years.  Henri  jean10 
has  reported  success  in  producing  tetanus  with  bacilli  from  a  splinter 
of  wood  infected  eleven  years  before. 

10  Henrijean,  Ann.  de  la  soc.  med.  Chir.  de  Liege,  1891. 


THE  ANAEROBIC   BACILLI  731 

Tetanus  Toxin. — The  pathogenicity  of  the  tetanus  bacillus 
depends  entirely  upon  the  soluble  toxin  which  it  produces.  This 
toxin  is  produced  in  suitable  media  by  all  strains  of  virulent  tetanus 
bacilli,  individual  strains  showing  less  variation  in  this  respect  than 
do  the  separate  strains  of  diphtheria  bacilli.  While  partial  aerobiosis 
does  not  completely  eliminate  toxin  formation,  anaerobic  conditions 
are  by  far  more  favorable  for  its  development. 

The  medium  most  frequently  employed  for  the  production  of 
tetanus  toxin  is  neutral  or  slightly  alkaline  beef-infusion  bouillon 
containing  five-tenths  per  cent  NaCl  and  one  per  cent  pepton.  Glu- 
cose, sodium  formate,  or  tincture  of  litmus  may  be  added,  but  while 
these  substances  increase  the  speed  of  growth  of  the  bacilli  they 
do  not  seem  to  enhance  the  degree  of  toxicity  of  the  cultures.  Glu- 
cose is  said  even  to  be  unfavorable  for  strong  toxin  development. 
It  is  important,  too,  that  the  bouillon  shall  be  freshly  prepared.11 
There  does  not  seem  to  be  any  direct  relationship  between  the 
amount  of  growth  and  the  degree  of  toxicity  of  the  cultures.  Under 
anaerobic  conditions  in  suitable  bouillon  and  grown  at  37.5°  C.,  the 
maximum  toxin  content  of  the  cultures  is  reached  in  from  ten  days 
to  two  weeks.  After  this  time  the  toxin  deteriorates  rapidly. 

Tetanus  toxin  has  been  produced  without  resort  to  mechanical 
anaerobic  methods  by  several  observers,  notably  by  Debrand,12  by 
cultivating  the  bacilli  in  bouillon  in  symbiosis  with  Bacillus  subtilis. 
By  this  method,  Debrand  claims  to  have  produced  toxin  which  was 
fully  as  potent  as  that  produced  by  anaerobic  cultivation. 

The  tetanus  toxin,  in  solution  in  the  bouillon  cultures,  may  be 
separated  from  the  bacteria  by  filtration  through  Berkefeld  or  Cham- 
berland  filters.  Since  the  poison  in  such  filtrates  deteriorates  very 
rapidly,  much  more  rapidly  even  than  diphtheria  toxin,  various 
methods  have  been  devised  to  obtain  the  toxin  in  the  solid  state. 
The  most  useful  of  these  is  precipitation  of  the  poison  out  of  solu- 
tion by  saturation  with  ammonium  sulphate.13  Very  little  of  the 
toxin  is  lost  by  this  method  and,  thoroughly  dried  and  stocked  in 
vacuum  tubes,  together  with  anhydrous  phosphoric  acid,  it  may  be 
preserved  indefinitely  without  deterioration.  The  precipitate  thus 
formed  is  easily  soluble  in  water  or  salt  solution,  and  therefore 


11  Vaillard  et  Vincent,  Ann.  de  Pinst.  Pasteur,  1891. 

12  Debrand,  Ann.  de  1'inst.  Pasteur,  1890,  1902. 

13  Brieger  und  Cohn,  Zeit.  f.  Hyg.,  xv. 


732  PATHOGENIC   MICROORGANISMS 

permits  of  the  preparation  of  uniform  solutions  for  purposes  of 
standardization. 

Brieger  and  Boer14  have  also  succeeded  in  precipitating  the  toxin 
out  of  broth  solution  with  zinc  chloride.  Vaillard  and  Vincent15 
have  procured  it  in  the  dry  state  by  evaporation  in  vacuo. 

Brieger  and  Cohn,16  Brieger  and  Boer,17  and  others  have  at- 
tempted to  isolate  tetanus  poison,  removing  the  proteins  from  the 
ammonium  sulphate  precipitate  by  various  chemical  methods.  The 
purest  preparations  obtained  have  been  in  the  form  of  fine  yellowish 
flakes,  soluble  in  water,  insoluble  in  alcohol  and  ether.  Solutions 
of  this  substance  have  failed  to  give  the  usual  protein  reactions. 

The  toxin  when  in  solution  is  extremely  sensitive  to  heat.  Kita- 
sato18  states  that  exposure  to  68°  C.  for  five  minutes  destroys  it 
completely.  Dry  toxin  is  more  resistant,19  often  withstanding  tem- 
peratures of  120°  C.  for  more  than  fifteen  minutes.  Exposure  to 
direct  sunlight  destroys  the  poison  in  fifteen  to  eighteen  hours.20 

Interesting  experiments  as  to  the  action  of  eosin  upon  tetanus 
toxin  have  been  carried  out  by  various  observers.  Flexner  and 
Noguchi21  found  that  five  per  cent  eosin  added  to  the  toxin  would 
destroy  it  within  one  hour.  This  action  is  ascribed  to  the  photo- 
dynamic  power  of  the  eosin. 

Tetanus  toxin  is  one  of  the  most  powerful  poisons  known  to  us. 
Filtrates  of  broth  cultures,  in  quantities  of  0.000,005  c.c.,  will  often 
prove  fatal  to  mice  of  ten  grams  weight.  Dry  toxin  obtained  by 
ammonium  sulphate  precipitation22  is  quantitatively  even  stronger, 
values  of  0.000,001  gram  as  a  lethal  dose  for  a  mouse  of  the  given 
weight  not  being  uncommon.  Brieger  and  Cohn23  succeeded  in  pro- 
ducing a  dry  toxin  capable  of  killing  mice  in  doses  of  0.000,000,05 
gram. 

Different  species  of  animals  show  great  variation  in  their  sus- 
ceptibility to  tetanus  toxin.  Human  beings  and  horses  are  probably 

14  Brieger  und  Boer,  Zeit.  f.  Hyg.  xxi. 

15  Vaillard  et  Vincent,  Ann.  de  1'inst.  Pasteur,  1891. 

16  Brieger  und  Cohn,  loc.  cit. 

17  Brieger  und  Boer,  Zeit.  f.  Hyg.,  xxi. 

18  Kitasato,  Zeit.  f .  Hyg.,  x. 

19  Morax  et  Marie,  Ann.  de  1'inst.  Pasteur,  1902. 

20  Fermi  und  Pernossi,  Cent,  f .  Bakt.,  xv. 

21  Flexner  and  Noguchi,  "Studies  from  Rockefeller  Inst.,"  v.,  1905. 

22  Brieger  und  Cohn.,  loc.  cit. 

*  Brieger  und  Cohn,  Zeit.  f .  Hyg.,  xv. 


THE  ANAEROBIC  BACILLI  733 

the  most  susceptible  species  in  proportion  to  their  body  weight.  The 
common  domestic  fowls  are  extremely  resistant.  Calculated  for 
grams  of  body  weight,  the  horse  is  twelve  times  as  susceptible  as 
the  mouse,  the  guinea-pig  six  times  as  susceptible  as  the  mouse. 
The  hen,  on  the  other  hand,  is  200,000  times  more  resistant  than  the 
mouse. 

After  the  inoculation  of  an  animal  with  tetanus  toxin  there  is 
always  a  definite  period  of  incubation  before  the  toxic  spasms  set 
in.  This  period  may  be  shortened  by  increase  of  the  dose,  but  never 
entirely  eliminated.24  When  the  toxin  is  injected  subcutaneously, 
spasms  begin  first  in  the  muscles  nearest  the  point  of  inoculation. 
Intravenous  inoculation,25  on  the  other  hand,  usually  results  in 
general  tetanus  of  all  the  muscles. .  The  feeding  of  toxin  does  not 
produce  disease,  the  poison  being  passed  through  the  bowel  un- 
altered. 

The  harmful  action  of  tetanus  toxin  is  generally  attributed  to 
its  affinity  for  the  central  nervous  system.  Wassermann  and  Takaki26 
show  that  tetanus  toxin  was  fully  neutralized  when  mixed  with 
brain  substance.  Other  organs — liver  and  spleen,  for  instance — 
showed  no  such  neutralizing  power.  The  central  origin  of  the  tetanic 
contractions  was  made  very  evident  by  the  work  of  Gumprecht,27 
who  succeeded  in  stopping  the  spasms  in  a  given  region  by  division 
of  the  supplying  motor  nerves. 

The  manner  in  which  the  toxin  reaches  the  central  nervous  system 
has  been  extensively  investigated,  chiefly  by  Meyer  and  Ransom, 
and  Marie  and  Morax.  Meyer  and  Ransom28  from  a  series  of  careful 
experiments  reached  the  conclusion  that  the  toxin  is  conducted  to 
the  nerve  centers  along  the  paths  of  the  motor  nerves.  Injected 
into  the  circulation,29  the  toxin  reaches  simultaneously  all  the  motor 
nerve  endings,  producing  general  tetanus.  In  this  case  too,  there- 
fore, the  poison  from  the  blood  can  not  pass  directly  into  the  central 
nervous  system,  but  must  follow  the  route  of  nerve  tracts. 

These  observations  have  been  of  great  practical  value  in  that  they 
pointed  to  the  desirability  of  the  injection  of  tetanus  antitoxin 

24  Courmont  et  Doyen,  Arch,  de  phys.,  1893. 

25  Ransom,  Deut.  med.  Woch.,  1893. 

26  Wassermann  und  Takaki,  Berl.  kiln.  Woch.,  1898. 

27  Gumprecht,  Pfluger's  Arch.,  1895. 

28  Meyer  und  Ransom,  Arch.,  f .  exp.  Pharm.  u.  Path.,  xlix. 

29  Marie  et  Morax,  Ann.  de  1'inst.  Pasteur,  1902. 


734  PATHOGENIC   MICROORGANISMS 

directly  into  the  nerves  and  the  central  nervous  system  in  active 
cases. 

Tetanolysin. — Tetanus  bouillon  contains,  besides  the  "tetano- 
spasmin ' '  described  xabove  which  produces  the  familiar  symptoms  of 
the  disease,  another  substance  discovered  by  Ehrlich30  and  named 
by  him  "tetanolysin. "  Tetanolysin  has  the  power  of  causing  liemol- 
ysis  of  the  red  blood  corpuscles  of  various  animals,  and  is  an  entire ly 
separate  substance  from  tetanospasmin.  It  may  be  removed  from 
toxic  broth  by  admixture  of  red  blood  cells,  is  more  thermolabile 
than  the  tetanospasmin,  and  gives  rise  to  an  antihemolysin  when 
injected  into  animals. 

Pathogenicity. — The  comparative  infrequency  of  tetanus  infection 
is  in  marked  contrast  to  the  wide  distribution  of  the  bacilli  in  nature. 
Introduced  into  the  animal  body  as  spores,  and  free-  from  toxin, 
they  may  often  fail  to  incite  disease,  easily  falling  prey  to  phagocy- 
tosis and  other  protective  agencies  before  the  vegetative  forms 
develop  and  toxin  is  formed.  The  protective  importance  of  phagocy- 
tosis was  demonstrated  by  Vaillard  and  Rouget,31  who  introduced 
tetanus  spores  inclosed  in  paper  sacs  into  the  animal  body.  By  the 
paper  capsules  the  spores  were  protected  from  the  leucocytes,  not 
from  the  body  fluids.  Nevertheless,  tetanus  developed  in  the  animals. 
The  nature  of  the  wound  and  the  simultaneous  presence  of  other 
microorganisms  seem  to  be  important  factors  in  determining  whether 
or  not  the  tetanus  bacilli  shall  be  enabled  to  proliferate.  Deep, 
lacerated  wounds,  in  which  there  has  been  considerable  tissue  de- 
struction, and  in  which  chips  of  glass,  wood  splinters,  or  grains  of 
dirt  have  become  embedded,  are  particularly  favorable  for  the 
development  of  these  germs.  The  injuries  of  compound  fractures 
and  of  gunshot  wounds  are  especially  liable  to  supply  these  condi- 
tions, and  the  presence  in  such  wounds  of  the  common  pus  cocci, 
or  of  other  more  harmless  parasites,  may  aid  materially  in  furnishing 
an  environment  suitable  for  the  growth  of  the  tetanus  bacilli.  Apart 
from  its  occurrence  following  trauma,  tetanus  has  been  not  infre- 
quently observed  after  childbirth,32  and  isolated  cases  have  been 
reported  in  which  it  has  followed  diphtheria  and  ulcerative  lesions 
of  the  throat.33 


30  Ehrlich,  Berlin,  kl.  Woch.,  1898. 

31  Vaillard  et  Rouget,  Ann.  de  1'inst.  Pasteur,  1892. 

32  Baginsky,  Deut.  med.  Woch.,  1893, 

,  Wien.  med.  Woch.,  1895. 


THE  ANAEROBIC  BACILLI  735 

A  definite  period  of  incubation  elapses  between  the  time  of  in- 
fection with  tetanus  bacilli  and  the  development  of  the  first  symp- 
toms. In  man  this  may  last  from  five  to  seven  days  in  acute  cases, 
to  from  four  to  five  weeks  in  the  more  chronic  ones.  Experimental 
inoculation  of  guinea-pigs  is  followed  usually  in  from  one  to  three 
days  by  rigidity  of  the  muscles  nearest  the  point  of  infection.  This 
spastic  condition  rapidly  extends  to  other  parts  and  finally  leads 
to  death,  which  occurs  within  four  or  five  days  after  infection. 

Autopsies  upon  human  beings  or  animals  dead  of  tetanus  reveal 
few  and  insignificant  lesions.  The  initial  point  of  infection,  if  at 
all  evident,  is  apt  to  be  small  and  innocent  in  appearance.  Further 
than  a  general  and  moderate  congestion,  the  organs  show  no 
pathological  changes.  Bacilli  are  found  sparsely  even  at  the  point 
of  infection,  and  have  been  but  rarely  demonstrated  in  the  blood 
or  viscera.  Nicolaier  succeeded  in  producing  tetanus  with  the  organs 
of  infected  animals  in  but  eleven  out  of  fifty-two  cases.  More  re- 
cently, Tizzoni34  and  Creite35  have  succeeded  in  cultivating  tetanus 
bacilli  out  of  the  spleen  and  heart 's  blood  of  infected  human  beings. 

The  researches  of  Tarozzi86  and  of  Canfora37  have  shown  also 
that  spores  may  be  transported  from  the  site  of  inoculation  to  the 
liver,  spleen,  and  other  organs,  and  there  lie  dormant  for  as  long 
as  fifty-one  days.  If  injury  of  the  organ  is  experimentally  practiced 
and  dead  tissue  or  blood  clot  produced,  the  spores  may  develop  and 
tetanus  ensue.  These  experiments  may  explain  cases  of  so-called 
crypto  genie  tetanus. 

In  man  tetanus  may  take  either  an  acute  or  chronic  form,  the 
word  "chronic"  here  meaning  simply  that  the  onset  is  less  abrupt, 
the  incubation  time  longer,  the  symptoms  slower  in  development  and 
the  prognosis  more  favorable.  In  the  acute  form,  the  incubation 
time  ranges  from  three  or  four  days  to  ten  or  fourteen  days,  the 
common  very  rapid  cases  taking  about  seven.  In  the  so-called 
chronic  form  the  incubation  time  may  occasionally  exceed  a  month. 
The  first  symptoms  usually  consist  in  headache  and  general  depres- 
sion; followed  rather  rapidly  by  difficulties  in  opening  the  mouth, 
due  to  spasms  or  trismus  of  the  masseters.  There  is  slight  stiffness 
of  the  neck  which  makes  it  difficult  for  the  patient  to  bring  the 

34  Tizzoni,  Ziegler's  Beit.,  vii. 

35  Creite,  Cent.  f.  Bakt.,  xxxvii. 

36  Tarozzi,  Cent.  f.  Bakt.  Orig.  xxxviii. 

37  Canfora,  Cent.  f.  Bakt.  Orig.  xlv. 


736  PATHOGENIC   MICROORGANISMS 

chin  forward  on  the  chest.  Gradually  there  develops  a  spasm  of 
the  muscles  of  the  cheeks  which  results  in  a  drawing  up  of  the 
tissues  about  the  mouth,  giving  a  curious  and  characteristic  expres- 
sion. Gradually  the  spasms  extend  to  the  trunk  and  back,  with 
the  development  of  opisthotonos  after  several  days.  Difficulty  in 
swallowing  may  ensue,  and  there  may  be  involuntary  movements 
of  urine  and  feces.  The  localization  of  the  symptoms  to  some 
extent  follows  the  location  of  the  injury.  Tetanus  may  occur  in 
the  new  born,  occasionally,  developing  soon  after  birth.  For  differ- 
ential diagnosis,  it  is  best  to  refer  to  books  on  general  medicine 
and  surgery. 

Many  types  of  atypical  tetanus  in  untreated  and  in  prophy- 
lactically  treated  cases  have  been  reported,  a  description  of  which 
can  be  found  in  extenso  in  the  volume  on  "The  Abnormal  Forms  of 
Tetanus"  by  Courtois-Suffit  and  Giroux  in  the  British  Medical  War 
Manuals,  published  in  1918.  They  speak  of  splanchnic  tetanus  char- 
acterized especially  by  the  involvement  of  the  muscles  of  deglutition 
and  respiration,  with  great  dysphagia.  Simple  cephalic  tetanus  in 
which  the  infection  may  be  confined  to  the  head,  is  a  type  in  which 
dysphagic  and  paralytic  symptoms  are  never  present,  and  which 
result  most  frequently  from  wounds  of  the  head.  It  may  be  char- 
acterized only  by  unilateral  and  bilateral  trismus,  or  by  contraction 
of  muscles  of  the  face.  There  is,  however,  a  dysphagic  form  of 
this  in  which  pharyngeal  spasms  precede  trismus.  Rarely  they  have 
noticed  a  so-called  hydrophobic  form  in  which  convulsions  accom- 
pany the  spasms. 

PROPHYLACTIC  USE  OF  TETANUS  ANTITOXIN 

The  most  important  use  for  tetanus  antitoxin  which  has  been 
found  hitherto,  is  its  prophylactic  administration.  The  methods  of 
applying  this  have  varied  in  different  parts  of  the  world  and  in 
different  armies.  That  it  is  of  great  value  was  demonstrated  by 
the  almost  immediate  reduction  of  tetanus  in  wounded  soldiers  after 
the  universal  introduction  of  prophylactic  tetanus  antitoxin  in  all 
the  armies  in  the  field.  The  wounds  which  are  particularly  danger- 
ous as  far  as  tetanus  is  concerned  are  those  in  which  there  is  con- 
siderable laceration,  especially  injury  to  bone,  and  in  which  dirt, 
and  especially  manured  soil  or  soil  from  cultivated  fields,  and  feces, 
are  likely  to  be  present.  The  growth  of  tetanus  bacilli  is  favored 
by  the  presence  of  dead  tissue  and  other  infected  organisms.  Studies 


THE  ANAEROBIC  BACILLI  737 

by  members  of  the  United  States  Public  Health  Service  have  shown 
that  tetanus  can  be  produced  with  regularity  if  staphylococcus  in- 
fection is  added  to  the  infection  with  tetanus  spores,  and  injury  of 
tissue  by  the  injection  of  small  quantities  of  such  substances  as 
quinine,  may  start  the  growth  of  latent  tetanus  spores  with  subse- 
quent development  of  the  disease.  Tetanus  spores  pass  through  the 
intestinal  canals  of  animals  and  man  without  injury,  and  are  dis- 
tributed in  the  soil  where  they  can  live  for  almost  unlimited  periods. 
Wounds  inflicted  upon  men  in  the  field,  especially  -by  the  blunt  and 
ragged  projectiles  of  high  explosives,  and  by  any  injury  passing 
through'  soil  and  filth  covered  clothing,  through  unwashed  skin, 
furnish  an  ideal  nidus  for  infection.  In  consequence,  in  prac- 
tically all  the  allied  armies  every  wounded  man  was  given  an 
injection  of  about  1,000  to  1,500  units  of  tetanus  antitoxin  as  soon 
after  the  injury  as  he  came  under  medical  observation.  In  civilian 
life,  the  wounds  that  require  similar  prophylactic  treatment  are 
those  inflected  with  much  traumatism  and  under  dirty  conditions, 
especially  those  in  which  compound  fractures  are  involved. 

We  refrain  from  giving  any  set  rules  for  prophylactic  treatment. 
The  principles  involved  are  that  the  injection  of  from  500  to  1,500 
or  even  up  to  5,000  units  should  be  made  subcutaneously  as  soon 
as  possible  after  the  injury.  It  should  be  remembered  that  the 
first  injection  may  not  be  sufficient.  The  antitoxin  gradually  dis- 
appears in  the  course  of  about  twelve  days,  and  wounds  that  are 
slow  in  cleaning  up  or  cases  in  which  secondary  interference,  such 
as  removal  of  sequestrum,  resetting  of  bones,  etc.  becomes  necessary, 
may  call  for  a  second  injection  after  six  to  eight  days,  with  due 
precautions  to  prevent  anaphylaxis.  In  such  cases,  according  to 
the  judgment  of  the  surgeon,  second  injections  should  become  almost 
the  rule  since  experience  in  the  war  has  shown  that  after  two 
injections  tetanus  is  very  rare  in  appearance. 

Recently,  very  important  advances  in  our  knowledge  of  tetanus 
have  been  made  by  W.  J.  Tulloch38  who  in  1917  showed  that  tetanus 
bacilli  could  be  classified  into  at  least  three  and  perhaps  four  types 
by  agglutination  with  anti-bacterial  serum  prepared  by  injection  of 
the  bodies  of  tetanus  bacilli.  The  important  point  which  arises  in 
this  connection  is,  of  course,  whether  the  toxins  produced  by  these 
various  agglutinative  types  differ  either  qualitatively  or  quantita- 

38  Tulloch,  Journal  of  the  R.  A.  M.  C.,  Dec.  1917. 


738  PATHOGENIC   MICROORGANISMS 

lively.  Subsequent  investigations  by  Tulloch39  showed  that  of  a 
large  number  of  isolations  from  infections  on  the  Western  Front, 
his  type  I  organisms  were  the  most  frequent,  type  II  and  type  III 
next,  and  type  IV  the  least  common.  It  seemed,  in  these  investiga- 
tions, that  the  agglutinin  titer  of  an  antitoxic  serum  is  no  index  to 
its  antitoxic  value;  the  agglutinating  sera,  even  when  of  very  high 
titer,  does  not  bring  out  group  reactions,  but  maintains  a  sharp 
separation  of  the  types  and  that  the  types  remain  true  even  after 
prolonged  cultivation.  The  types  could  be  demonstrated  by  opsonic 
as  well  as  by  agglutination  experiments.  Most  important  is  the 
observation  that  the  spasm-producing  toxin  is  not  specific  to  the 
types,  although  there  may  be  quantitative  differences  in  toxin 
production.  Tulloch 's  investigations  suggest  that  anti-bacterial 
properties  in  tetanus  sera,  if  polyvalent,  would  aid  considerably  in 
the  phagocytosis  of  the  organisms,  and,  therefore,  have  a  prophy- 
lactic and  therapeutic  value.  His  work,  also,  seems  to  indicate  that 
small  amounts  of  the  tetanospasmin  do  not  locally  aid  the  growth 
of  the  tetanus  bacillus  to  any  considerable  extent,  an  observation 
in  distinct  contrast  with  the  earlier  work  on  this  subject.  However, 
B.  Welchii  toxin  and,  to  a  smaller  extent,  that  of  Vibrion  Septique, 
considerably  aid  the  growth  of  tetanus,  by,  as  he  expresses  it, 
"devitalizing"  the  tissues.  This  leads  Tulloch  to  favor  a  procedure, 
advised  by  other  observers  on  purely  bacteriological  grounds, 
namely  the  combining  of  antitoxins  against  the  poisons  of  B.  Welchii 
and  of  Vibrion  Septique  with  Tetanus  antitoxin,  in  the  prophylactic 
treatment  of  war  wounds. 

Even  when  this  is  done,  however,  he  cautions  against  any  feeling 
of  false  security  which  might  lead  to  the  neglect  of  surgical  prophy- 
laxis. 

He  emphasizes  the  great  importance  of  early  excision  of  the 
wound  area.  No  particular  dressings  seem  to  make  a  great  deal 
of  difference,  but  thorough  excision  seems  to  have  considerable  influ- 
ence on  the  development  of  tetanus  and  on  the  mortality. 

The  Treatment  of  Developed  Tetanus.— To  speak  of  the  specific 
treatment  of  tetanus  without  saying  a  few  words  about  the  surgical 
treatment  would  be  taking  the  risk  of  conveying  a  false  impression. 
Therefore,  though  our  business  here  is  concerned  largely  with  specific 
treatment,  we  wish  to  emphasize  that  surgical  treatment  must  al- 


89  Tulloch,  Journal  of  Hygiene,  vol.  18,  1919,  p.  103. 


THE  ANAEROBIC  BACILLI 


739 


ways  be  carried  out  whatever  method  of  serum  therapy  be  employed. 
This  must  consist  in  thoroughly  cleansing  the  wound,  removal  of 
foreign  bodies,  fragments  of  projectiles,  clothing,  gross  dirt,  etc., 
and,  as  the  late  war  has  shown,  it  is  perhaps  best  whenever  possible 
to  carry  out  debridement  or  excision  of  the  wound.  From  Tulloch  's40 
studies  it  would  appear  that  no  dressing  is  particularly  superior 
to  any  other,  and  we  doubt  very  much  whether  oxidizing  agents, 
like  the  insufflation  of  oxygen,  peroxides,  etc.,  are  of  much  use, 
because  of  the  reducing  powers  of  tissues. 

As  to  specific  serum  treatment,  it  must  be  admitted  that  earlier 
results  were  very  disappointing,  and  the  mere  subcutaneous  injec- 
tion even  of  large  doses  of  tetanus  antitoxin  has  usually  been  dis- 
appointing in  the  acute  forms  of  the  disease.  This  has  perhaps  been 
largely  due  to  the  fact  that  the  injected  antitoxin  could  have  no 
possible  influence  on  the  toxin  which  had  already  become  united 
with  the  substances  of  the  nerve  tissues.  A  great  many  modifications 
in  the  method  of  injection  have  been  employed,  such  as  injection 
directly  into  the  central  nervous  system  and  into  the  nerve  trunks, 
themselves.  It  may  be  stated  that  the  relative  acuteness  of  the 
tetanus  infection  very  definitely  influences  the  results  of  serum 
therapy.  The  following  table  taken  from  Etienne  and  copied  from 
Courtois-Suffit  and  Giroux  in  the  series  of  British  War  Manuals 
mentioned  above,  gives  a  general  idea  of  the  usefulness  of  serum 
therapy  in  this  disease: 


Mortality 

before  the 

Incubation. 

Recoveries. 

Deaths. 

Mortality. 

Introduction  of 
Serotherapy, 

according  to 

Brunner. 

1  to  5  days  

3 

7 

70% 

90% 

5  to  10  days 

20 

7 

29% 

70% 

10  to  12  days  

7 

1 

13  3% 

Over  12  days  . 

15 

1 

6  6% 

This  table  cited  from  Courtois-Suffit  and  Giroux,  Military  Medical  Manuals, 
Univ.  of  London  Press,  London,  1918,  page  193. 

It  would  be  useless  to  go  into  the  various  methods  of  adminis- 
tering tetanus  antitoxin  that  have  been  tried,  and  we  will  confine 


40  Tulloch,  Jour.  R.  A.  M.  C.,  December,  1917,  Jour.  Hygiene,  18,  1919,  103. 


740  PATHOGENIC   MICROORGANISMS 

ourselves  to  the  intraspinal  method  developed  in  this  country  by 
Park  and  Nicoll,41  and  in  France  by  Doyen,  and  gradually  coming 
into  general  use.  As  advised  by  Park  and  Nicoll,  a  spinal  puncture 
is  made  and  a  moderate  amount  of  spinal  fluid  taken  out.  Then, 
slowly  by  gravity  from  3,000  to  5,000  units  of  tetanus  antitoxin  are 
injected  until  a  total  amount  of  3  c.c.  has  been  reached,  the  amount 
injected  approximately  replacing  the  amount  of  fluid  withdrawn. 
At  the  same  time,  10,000  units  are  given  intravenously  or  intramus- 
cularly. This  procedure  must  be  repeated  according  to  indications. 
For  other  forms  of  treatment  such  as  carbolic  acid  injections, 
magnesium  sulphate,  etc.,  we  must  refer  the  reader  to  books  on 
surgical  therapy. 

BACILLUS  BOTULINUS 

Meat  poisoning  was  formerly  regarded  as  entirely  dependent 
upon  putrefactive  changes  in  infected  meat,  resulting  in  the 
production  of  ptomains  or  other  harmful  products  of  bacterial 
putrefaction.  It  was  not  until  1888  that  certain  of  these  cases  were 
definitely  recognized  as  true  bacterial  infections,  in  which  the  pre- 
formed poison  probably  aided  only  in  establishing  the  infection. 
Gartner,  in  that  year,  discovered  the  Bacillus  enteritidis,  a  micro- 
organism belonging  to  the  group  of  the  paratyphoid  bacilli,  and  demon- 
strated its  presence  both  in  the  infecting  meat  and  in  the  intestinal 
tracts  of  patients.  The  characteristics  of  this  type  of  meat  poisoning 
have  been  discussed  more  particularly  in  the  section  describing  the 
bacillus  of  Gartner  and  its  allied  forms. 

There  is  another  type  of  meat  poisoning,  however,  which  is  not 
only  much  more  severe,  but  is  characterized  by  a  clinical  picture 
more  significant  of  a  profound  systemic  toxemia  than  of  a  mere 
gastroenteric  irritation.  The  etiological  factor  underlying  this  type 
of  infection  was  first  demonstrated  by  Van  Ermengem,42  in  1896, 
and  named  Bacillus  botulinus.  Van  Ermengem  isolated  the  bacillus 
from  a  pickled  ham,  the  ingestion  of  which  had  caused  disease  in 
a  large  number  of  persons.  Of  the  thirty-four  individuals  who  had 
eaten  of  it,  all  were  attacked,  about  ten  of  them  very  severely.  Van 
Ermengem  found  the  bacilli  in  large  numbers  lying  between  the 
muscle  fibers  in  the  ham,  and  was  able  to  cultivate  the  same  micro- 

41  Park  and  Nicoll,  Jour.  A.  M.  A.,  63,  1914,  245. 

42  Van  Ermengem,  Cent,  f,  Bakt.,  xix,  1896;  Zeit.  f,  Hyg.,  xxvi,  1897. 


THE  ANAEROBIC  BACILLI  741 

organism  from  the  stomach  and  spleen  of  one  of  those  who  died 
of  the  infection.  The  results  of  Van  Ermengem  have  been  confirmed 
by  Romer,43  and  others. 

Morphology  and  Staining. — Bacillus  botulinus  is  a  large,  straight 
rod  with  rounded  ends,  4  to  6  micra  in  length  by  0.9  to  1.2  micra 
in  thickness.  The  bacilli  are  either  single  or  grouped  in  very  short 
chains.  Involution  forms  are  numerous  on  artificial  media.  The 
bacillus  is  slightly  motile  and  possesses  from  four  to  eight  undulated 
flagella,  peripherally  arranged.  Spores  are  formed  in  suitable  media, 
most  regularly  in  glucose-gelatin  of  a  distinctly  alkaline  titer.  The 
spores  are  oval  and  usually  situated  near  the  end  of  the  bacillus, 
rarely  in  its  center.  Spores  are  formed  most  abundantly  when 
cultivation  is  carried  out  at  20°  to  25°  C.,  and  are  usually  absent 
when  higher  temperatures  are  employed. 

The  bacillus  is  easily  stained  by  the  usual  aqueous  anilin  dyes, 
and  retains  the  anilm-gentian-violet  when  stained  by  Gram.  It  is 
necessary,  however,  in  carrying  out  Gram's  stain  to  decolorize  care- 
fully with  alcohol  since  overdecolorization  is  easily  accomplished, 
leaving  the  result  doubtful. 

Cultivation. — The  bacillus  is  a  strict  anaerobe.  In  anaerobic  en- 
vironment it  is  easily  cultivated  on  the  usual  meat-infusion  media. 
It  grows  most  readily  at  temperatures  about  25°  C.,  less  luxuriantly 
at  temperatures  of  35°  C.  and  over. 

The  bacillus  is  delicately  susceptible  to  the  reaction  of  media, 
growing  only  in  those  which  are  neutral  or  moderately  alkaline. 

In  deep  stab  cultures  in  one  per  cent  glucose  agar,  growth  is  at  first 
noticed  as  a  thin,  white  column,  not  reaching  to  the  surface  of  the 
medium.  Soon  the  medium  is  cracked  and  split  by  the  abundant 
formation  of  gas.  On  agar  plates,  the  colonies  are  yellowish, 
opalescent,  and  round,  and  show  a  finely  fringed  periphery. 

On  gelatin,  at  20°  to  25°  C.,  growth  is  rapid  and  abundant,  and 
differs  little  from  that  on  agar,  except  that,  besides  the  formation 
of  gas,  there  is  energetic  fluidification  of  the  medium.  On  glucose- 
gelatin  plates,  Van  Ermengem  describes  the  colonies  as  round,  yel- 
lowish, transparent,  and  composed  of  coarse  granules  which,  along 
the  periphery  in  the  zone  of  fluidification,  show  constant  motion. 
The  appearance  of  the  surface  colonies  on  glucose-gelatin  plates  is 
regarded  by  the  discoverer  as  diagnostically  characteristic. 

43  Romer,  Cent,  f .  Bakt.  xxvii,  1900. 


742  PATHOGENIC   MICROORGANISMS 

In  glucose  Irotli  there  is  general  clouding  and  large  quantities  of 
gas  are  formed.  At  35°  C.  and  over,  the  gas  formation  ceases  after 
four  or  five  days,  the  broth  becoming  clear  with  a  yellowish-white 
flocculent  sediment.  At  lower  temperatures  this  does  not  occur. 

Milk  is  not  coagulated  and  disaccharids  and  polysaccharids  are  not 
fermented. 

The  gas  formed  in  cultures  consists  chiefly  of  hydrogen  and 
methane.  All  cultures  have  a  sour  odor,  like  butyric  acid,  but  this 
is  not  so  offensive  as  that  of  some  of  the  other  anaerobic  organisms. 

The  bacillus  lives  longest  in  gelatin  cultures,  but  even  upon  these, 
transplantations  should  be  done  every  four  to  six  weeks,  since  the 
spores  of  this  bacillus  show  less  viability  and  resistance  than  do 
those  of  most  spore-formers. 

Isolation. — The  isolation  of  B.  botulinus  from  infected  material 
is  often  quite  easy,  since  as  Burke  states,  few  other  organisms  may 
be  present  in  the  canned  or  pickled  food  products.  Often  anaerobic 
shake  cultures  in  agar  made  directly,  serve  to  isolate  the  organisms. 
In  most  contaminated  material,  however,  she  recommends  inoculation 
from  the  original  material  into  Von  Ermengem's  broth,  inoculating 
quite  richly,  duplicate  cultures  being  made  and  heated  at  60°  for 
one  hour,  to  destroy  non-spore  bearers.  These  cultures  are  then 
incubated  at  28°  after  which  parts  of  them  are  filtered,  and  the 
filtrate  in  quantities  of  1  c.c.  injected  into  250-gram  guinea-pigs. 
If  the  guinea-pigs  die  within  four  days,  the  pigs  are  again  tested 
with  specific  botulismus  antitoxin.  The  presence  of  the  organisms 
having  been  thus  proven  in  the  broth,  isolation  is  now  carried 
out  by  careful  anaerobic  plating  or  shake  cultures.  We  do  not  know 
whether  the  platinized  asbestos  method  has  been  used  in  botulismus 
work,  but  would  recommend  its  use  in  cases,  such  as  those  men- 
tioned. 

Botulismus  Toxin. — Botulismus  toxin  is  produced  under  condi- 
tions of  strict  anaerobiosis  on  any  media  on  which  the  organism 
will  grow  readily.  According  to  Dickson44  the  toxin  is  much  more 
potent  if  the  organisms  are  grown  on  an  alkalin  medium  and  in 
the  dark.  Von  Ermengem45  obtained  his  best  toxin  by  growing  the 
organisms  on  a  beef  infusion  broth  to  which  he  added  1  per  cent 

44  Dickson,  Monograph  of  the  Rock.  Inst.,  No.  8,  July  31,  1918,  (la).     Jour,  of 
the  A.  M.  A.,  65,  1915,  492. 

45  Von  Ermengem,  quoted  from  Kolle  and  Wassermann,  Second  Edition. 


THE  ANAEROBIC  BACILLI  743 

sodium  chloride,  1  per  cent  pepton  and  2  per  cent  glucose.  Leuchs46 
used  a  pork  infusion  with  0.5  per  cent  sodium  chlorid,  1  per  cent 
glucose  and  1  per  cent  pepton.  Landmann47  claimed  that  animal 
protein  was  necessary  for  good  toxin  production.  According  to 
Dickson  this  is  not  essential.  He  has  produced  toxin  in  media 
made  from  string  beans  and  from  peas,  and  found  that,  although 
an  alkalin  reaction  is  favorable,  an  acid  reaction  does  not  prevent 
toxin  formation.  According  to  Burke48  toxin  is  produced  as  readily 
at  37.5°  as  it  is  at  28°  C.  The  toxin  is  destroyed  at  temperatures 
of  about  80°.  Thorn,  Edmonson  and  Giltner49  claim  that  their 
toxin  was  destroyed  by  ten  minutes'  heating  at  75°  C.  Von  Ermen- 
gem's  original  report  was  that  heating  at  56°  for  three  hours  killed 
it,  as  does  heating  at  80°  for  one-half  hour.  According  to  Dickson, 
it  is  rapidly  destroyed  by  exposure  to  sunlight  and  to  air,  but  will 
maintain  its  virulence  for  six  months  if  kept  in  the  dark  as  it  would 
be  in  preserved  foods.  It  is  not  affected  by  drying  and  is  insoluble 
in  alcohol,  ether  and  chloroform.  Normal  soda,  20  per  cent  by 
volume,  is  stated  by  Dickson  to  destroy  it,  though  similar  amounts 
of  acid  do  not  reduce  its  virulence  in  twenty-four  hours. 

Its  potency  is  considerable.  Dickson  produced  his  strongest 
toxin  in  pork  and  beef  infusions,  but  also  obtained  potent  prepara- 
tions in  media  of  string  beans,  peas,  green  corn,  and  less  virulent 
toxin  in  media  prepared  from  asparagus,  artichokes,  peaches,  and 
apricots.  Brieger  and  Kempner50  obtained  a  toxin  of  which  0.000,001 
of  a  c.c.  would  kill  a  250-gram  guinea-pig  in  four  days,  and  (we 
quote  from  Dickson),  Von  Ermeiigem  found  in  one  of  his  outbreaks 
that  200  grams  of  the  poisonous  ham  caused  the  death  of  one  patient. 
He  quotes  another  case  in  which  a  piece  of  preserved  duck  the  size 
of  a  walnut  was  sufficient  to  cause  a  disease  lasting  six  weeks,  and 
in  his  own  series,  a  patient  died  after  tasting  a  small  spoonful  of 
spoiled  corn,  another  died  after  "  nibbling  a  portion  of  a  pod 
of  the  spoiled  string  beans."  A  third  was  quite  ill  after  tasting, 
but  not  swallowing  a  pod  of  beans.  An  important  point  is  the  claim 
that  has  been  made  by  Von  Ermengem  and  others  that  the  organism 
will  not  produce  toxin  in  the  tissues.  Injection  of  the  bacilli  alone 

46  Leuchs,  Zeit.  f.  Hyg.,  1910,  65,  55. 

47  Landmann,  cited  from  Dickson,  loc.  cit. 

48  Burke,  Jour.  Bacter.,  4,  1919,  555. 

49  Thorn,  Edmonson  and  Giltner,  Jour.  A.  M.  A..  Vol.  73,  1919,  page  901. 

50  Brieger  and  Kempner,  Deut.  med.  Woch.,  23,  1897,  521. 


744  PATHOGENIC   MICROORGANISMS 

into  suitable  animals  produces  no  botulismus,  and  intravenous  injec- 
tion or  feeding  of  the  bacilli  alone,  may  not  produce  symptoms. 
The  conclusion  drawn  by  Von  Ermengem  was  that  the  toxin  could 
not  be  produced  in  the  bodies  of  mammals. 

The  toxin  is  potent  for  monkeys,  rabbits,  guinea-pigs,  cats,  and 
various  birds.  Dickson  found  chickens  highly  susceptible,  and  also 
found  that  dogs  were  not  as  resistant  as  formerly  thought  to  be. 
The  most  susceptible  animals  seem  to  be  mice,  guinea-pigs  and 
monkeys ;  rabbits,  cats,  dogs  and  rats  are  relatively  resistant. 

In  studying  various  isolations  of  the  Bacillus;  Dickson  found  that 
the  strains  isolated  by  him  were  not  entirely  homologous,  and  that 
the  toxins  produced  by  some  of  them  were  not  neutralized  by  the 
antitoxin  produced  with  others.  Burke  also  found  two  types  that 
produced  heterologous  toxin  and  claims  that  the  strains  could  be  easily 
identified  by  a  toxin-antitoxin  test. 

Pathology. — In  animals,  according  to  Von  Ermengem  there  is  a 
general  hyperemia  of  the  organs  and  especially  of  the  nervous 
system.  Dickson  has  made  a  thorough  review  of  the  pathological 
work  done  by  Von  Ermengem,  Vander  Stricht,*  Marinesco51  and 
others,52  summarizing  the  pathology  as  follows:  In  the  central 
nervous  system  the  meninges  at  the  base  of  the  brain,  especially 
around  the  pons  and  the  medulla,  are  usually  more  markedly  con- 
gested than  at  the  cortex,  and  there  may  be  hemorrhage  in  the 
upper  part  of  the  cord  and  at  the  base  of  the  brain.  The  lungs  may 
be  hyperemic,  heart  muscles  flabby,  but  nothing  characteristic.  An 
important  and  regular  lesion  found  by  Ophuls53  was  multiple  throm- 
bosis in  both  the  arteries  and  veins  of  the  central  nervous  system. 
Ophuls  believes  this  is  due  to  a  certain  vasodilation  with  slowing 
of  the  blood  stream  due  to  a  powerful  paralyzing  effect  of  the  poison 
on  the  unstriped  muscles.  The  thromboses  are  particularly  common 
at  the  base  of  the  brain.  Ophuls,  too,  differs  from  others  in  believing 
that  the  specific  action  of  the  toxin  on  the  nerve  cells  themselves 
has  been  very  much  exaggerated  in  that  his  histological  examina- 
tions of  the  brains  of  fatal  cases  did  not  bear  out  this  earlier  opinion. 

Transmission  and  Occurrence  of  Botulismus, — Kempner  and  Pol- 


*  Stricht,  Quoted  from  Van  Ermengem,  in  Kolle  and  Wassermann. 
51  Marinesco,  Compt.  rend,  de  1'acad.  des  sci.,  Nov.,  1896. 
62  Kempner  und  Pollack,  Deut.  med.  Woch.,  xxxii,  1897. 

63  Ophuls,  with  Wilbur,  Arch.  Int.  Med.,  14,  1914,  589. 


THE  ANAEROBIC  BACILLI  745 

lack54  in  1897  isolated  B.  botulismus  from  the  intestines  of  a  normal 
hog,  but  Dickson  was  unable  to  find  the  organism  in  the  intestinal 
canals  of  250  grain-fed  hogs  in  the  slaughter  houses  of  San  Fran- 
cisco. He  also  collected  soil  from  gardens,  but  in  a  considerable 
series  of  specimens  did  not  find  organisms.  Burke55  examined  ma- 
terials from  many  different  localities  in  California  where  a  num- 
ber of  epidemics  had  occurred,  cultivating  from  large  varieties  of 
substances,  such  as  water,  hay,  vegetables,  fruits,  manure  from 
horses,  hogs,  chickens,  etc.  She  obtained  seven  cultures  of  the  B. 
botulinus  from  the  following  sources:  Moldy  cherries,  leaves  touched 
with  droppings  of  insects,  sprouts  from  bush-bean  plants,  manured 
bush  beans,  manure  from  a  hog,  and  moldy  hay.  These,  and  other 
investigations  seem  to  indicate  that  the  B.  of  botulismus  is  quite 
common  in  nature,  and  may  be  present  in  the  intestines  of  domestic 
animals,  such  as  hogs.  It  may  possibly  be  disseminated  by  insects, 
and  may  often  be  present  on  vegetables  and  fruits  at  the  time  that 
they  are  picked  for  canning. 

The  outbreaks  first  reported  were  largely  due  to  meat  and  it  is 
interesting  to  note  that  Van  Ermengem's  first  isolations  were  from 
pickled  meat,  thus  showing  that  ordinary  salting  or  brine  preserva- 
tion does  not  kill  the  botulinus  spores.  The  epidemics  that  have 
occurred  are  summarized  in  a  monograph  of  Mayer,56  and  more 
recently  in  an  admirable  study  of  the  disease  by  Dickson,  published 
in  a  Monograph  of  the  Rockefeller  Institute,57  and  carried  out  at 
the  Stanford  University  Pathological  Laboratory.  At  the  time  of 
Mayer's  publication  (in  1913 ),  800  European  cases  had  been 
observed  since  1882,  200  of  which  had  been  fatal.  Meat  in  general, 
or  animal  food  had  been  the  source  of  the  disease ;  and  the  generally 
prevalent  idea  at  that  time  was  that  meat  was  the  chief  source  of 
danger.  During  recent  years,  however,  the  studies  of  Dickson  and 
others  have  shown  that  vegetable  foods,  especially  canned  vegetables 
were  equally  as  dangerous.  Of  sixty-four  cases  collected  by  Dickson 
for  the  United  States  during  the  past  twenty-five  years,  fifty-four 
occurred  in  California.  Wilbur  and  Ophuls58  reported  an  outbreak 
in  1914  due  to  the  eating  of  canned  beans.  Since  that  time,  a 

54  Kempner  and  Pollack,  Deut.  med.  Woch.,  23,  1897,  505. 

55  Burke,  Jour.  Bacter.,  4,  1919,  541. 

56  Mayer,  Deut.  Vrtjschr.  off.  Gsndhtspflg.,  35,  1913,  8. 

57  Dickson,  Monograph  of  the  Rock.  Inst.,  No.  8,  July  31,  1918. 
68  Wilbur  and  Ophuls,  Arch.  Int.  Med.,  14,  1914,  589. 


746  PATHOGENIC   MICROORGANISMS 

number  of  other  cases  have  occurred  in  California  and  the  rest  of 
the  United  States,  many  of  which  have  been  studied  by  Dickson 
and  his  associates,  most  of  them  originating  from  canned  corn  and 
string  beans.  Thorn,  Edmonson  and  Giltner,  reported  an  outbreak 
occurred  in  1919,  traceable  to  canned  asparagus. 

Armstrong59  carefully  studied  an  outbreak  in  1919  which  was 
traced  to  ripe  olives.  It  is  impossible  in  this  space  to  do  justice 
to  the  large  and  valuable  botulismus  literature  which  has  developed 
during  the  last  few  years,  since  the  studies  of  Dickson  and  others 
have  renewed  the  interest  of  laboratory  workers  in  the  disease. 
The  mortality  of  the  disease  has  been  high,  and  for  the  United  States 
generally,  as  stated  by  Dickson,  it  has  been  over  64  per  cent. 

Attention  has  also  been  recently  called  to  the  relationship  of 
botulismus  in  man  to  the  disease  called  limber  neck  in  chickens. 
In  Hillsboro,  Oregon,  Dickson  states60  that  fifty  chickens  came  down 
after  eating  home-canned  corn  which  had  caused  the  death  of  a 
woman  who  tasted  it.  In  Hornbrook,  California,  between  fifty  and 
one  hundred  chickens  became  paralyzed  and  died  at  the  same  time 
as  the  woman  who  cared  for  them  died  of  "bulbar  paralysis."  In 
the  San  Jose  district,  eight  chickens  died  after  eating  home-canned 
string  beans  which  had  caused  the  death  of  a  woman;  and  seven 
chickens  died  in  Fallbrook,  California,  after  eating  home-canned 
apricots  which  had  killed  five  people.  Dickson  obtained  the  carcasses 
of  some  of  the  chickens  of  the  San  Jose  and  Hillsboro  outbreaks, 
and  from  the  gizzard  of  one  of  those  which  had  eaten  the  canned 
corn  and  from  the  crop  of  three  which  had  eaten  canned  beans,  he 
obtained  B.  Botulinus.  He  also  succeeded  in  producing  symptoms 
by  feeding  chickens  with  the  infected  material  obtained  in  this  way. 
He  described  the  symptoms  as  follows:  The  chickens  refuse  to  eat, 
remaining  quiet,  and  gradually  develop  weakness  of  the  neck,  wings 
and  legs,  finally  drooping  completely.  In  the  experimental  cases 
death  occurred  within  twenty-four  hours  after  feeding.  This,  apart 
from  its  diagnostic  value  and  experimental  importance,  may  have 
considerable  bearing  on  epidemiological  studies;  and  limber  neck  in 
chickens  should  always  be  inquired  into  when  human  cases  are 
observed  or  suspected. 

Clinical  Manifestations  of  Botulismus. — Botulismus  is  character- 
istic in  its  clinical  manifestations  and  should  not  present  great 

59  Armstrong,  Pub.  Health  Reports,  1919,  54,  December. 

60  Dickson,  Jour.  Amer.  Vet.  Assoc.,  January,  1917. 


THE  ANAEROBIC   BACILLI  747 

diagnostic  difficulties,  once  the  disease  is  suspected.  Since  the  toxin 
is  preformed  before  ingestion,  the  symptoms  are  not  long  in  follow- 
ing the  eating  of  infected  food,  coming  on  usually  within  twenty-four 
hours  or  less.  Delays  of  two  or  three  days,  however,  may  occur, 
and  should  not  throw  out  possible  positive  diagnosis.  The  earliest 
symptoms  usually  consist  in  a  general  weakness  and  lassitude,  with 
fatigue  and  some  headache.  Characteristic  is  the  frequent  lack  of 
any  symptoms  pointing  to  the  gastrointestinal  canal.  Constipation 
is  the  rule.  Very  early  in  the  disease,  disturbances  of  vision  may 
occur  which  are  due  to  impairment  of  the  muscles  of  the  eye-ball. 
There  is,  particularly,  involvement  of  the  third  cranial  nerve,  with 
blepharoptosis,  mydriasis,  impaired  light  reflex  and  diplopia.  There 
may  be  photophobia.  For  a  detailed  discussion  of  the  symptoma- 
tology, the  reader  is  referred  to  the  Monograph  of  Dickson.61  Im- 
pairment of  the  pharyngeal  muscles  may  produce  difficulties  in 
swallowing  with  inability  to  chew,  and  sluggishness  of  the  tongue 
with  thickness  of  speech.  Absence  of  temperature  is  an  important 
feature,  and  in  the  early  stages  there  is  usually  no  fever  and  no 
change  in  the  heart  rate.  Fatal  cases  usually  end  in  death  within 
three  to  seven  days,  due  either  to  cardiac  failure  or  terminal 
asphyxia.  In  discussing  the  differential  diagnosis,  Dickson  mentions 
particularly,  poliomyelitis,  cerebrospinal  syphilis,  early  stages  of 
bulbar  paralysis,  belladonna  poisoning  and  methyl  alcohol  poisoning. 
The  last  must  be  particularly  thought  of  during  the  present  period 
of  drought. 

Specific  Therapy. — Potent  antitoxins  may  be  produced  by  the 
treatment  of  susceptible  animals  with  toxin.  Kempner62  in  1897 
was  the  first  to  experiment  on  this  extensively,  using  the  Von 
Ermengem  strain,  and  producing  antitoxin  in  goats.  The  immuniza- 
tion of  small  laboratory  animals  is  comparatively  difficult,  unless 
minute  doses  and  attenuated  toxin  is  used.  The  chief  studies  on 
these  phases  of  the  problem  have  been  made  by  Forssman  and 
Lunstrom63  and  by  Leuchs.64  More  recently,  Dickson  and  Howitt65 
have  produced  potent  antitoxins  in  goats,  though  their  products, 
they  state,  were  not  as  powerful  as  those  reported  by  Kempner  and 


61  Dickxon,  Monograph  of  the  Rock.  Inst.,  No.  8,  July  31,  1918. 

82  Kempner,  Zeit.  f.  Hyg.,  26,  1897,  481. 

83  Foreman  and  Limstrom,  Ann.  do  I' Inst.  Past.,  16,  1902,  294. 

64  Leiichs,  Kollc  and  Wasserrnann  Handb.,  Second  Edition,  4,  939. 
85  Dickson  and  Howitt,  Jour.  A.  M.  A.,  74,  1919,  718. 


748  .  PATHOGENIC   MICROORGANISMS 

some  other  observers.  It  is  very  important  to  note  that  in  these 
experiments  antitoxin  produced  against  one  series  of  strains  had 
no  appreciable  effects  upon  the  toxins  of  three  other  strains.  This 
brings  out  the  great  importance  of  producing  curative  sera  by  the 
use  of  toxins  from  a  number  of  different  strains.  According  to 
Dickson,  there  are  at  least  two  types  which  must  be  used.  As  to 
the  therapeutic  value,  reports  have  been  confusing.  Dickson  advises 
intravenous  injection  and  states  that  his  procedure  would  be  as 
follows:  The  usually  precautions  for  the  administration  of  horse 
serum  should  be  observed,  and  the  patient  tested  for  skin  sensitive- 
ness. If  no  such  sensitiveness  is  found,  the  serum  should  be  injected 
immediately  into  a  vein  at  the  rate  of  not  more  than  1  c.c.  a  minute. 
Comparatively  large  doses  should  be  given,  since  the  amount  of 
toxin  ingested  may  be  quite  large. 

Prevention  of  Botulismus. — Deducing  preventive  measures  from 
the  facts  cited  in  the  above  paragraphs,  it  would  seem  that,  in  the 
first  place,  all  people  in  the  habit  of  preparing  canned  food  should 
be  thoroughly  alive  to  the  possibilities  of  contamination  and  know 
that  B.  botulinus  spores  may  be  present  on  fruit,  vegetables,  etc., 
before  they  are  preserved.  It  should  be  well  understood  that  food 
may  be  contaminated  with  botulinus,  without  being  changed  in 
any  way  in  its  gross  appearance,  and  that  not  even  the  slightest 
rancid  odor  which  sometimes  indicates  its  presence,  need  be  apparent. 
The  sterilization  of  canned  food,  sausages,  preserved  meat,  etc., 
should  be  thoroughly  attended  to  and  no  home-canned  preparations 
be  eaten  under  any  circumstances  unless  cooked  before  eating. 


CHAPTER   XXXVII 

THE  ANAEROBIC  BACILLI  (Continued) 

THE  ANAEROBIC  ORGANISMS  ASSOCIATED  WITH  TRAU- 
MATIC INJURIES,  WITH  A  CONSIDERATION  OF  THEIR 
IMPORTANCE  IN  WAR  SURGERY,1  ALSO  BACILLUS  OF 
SYMPTOMATIC  ANTHRAX 

THE  anaerobic  bacilli  which  infect  wounds  have  been  studied 
extensively  during  the  last  war,  and  the  literature  which  has  ap- 
peared on  this  subject  since  1914,  is  voluminous'.  Unfortunately 
much  that  has  been  written  is  inaccurate,  due  to  the  fact  that  in 
many  instances  the  work  was  carried  on  in  poorly  equipped  labora- 
tories and  under  difficulties.  The  two  most  important  sources  of 
confusion  in  this  field  are:  the  nomenclature  and  the  impurity  of 
cultures.  During  the  war  many  previously  described  bacilli  were 
rediscovered  and  given  new  names,  and  the  literature  is  full  of 
papers  claiming  or  disclaiming  that  certain  organisms  isolated  from 
wounds  are  identical  with  organisms  described  by  earlier  workers. 
Even  such  a  well-known  species  as  B.  Welchii  appears  in  the  litera- 
ture under  four  names. 

It  is  now  generally  considered  that  in  all  probability  most  of 
the  early  descriptions  of  anaerobic  bacilli  cannot  be  relied  upon, 
because  of  the  extreme  difficulty  of  isolating  the  anaerobes  in  pure 
culture.  Many  investigators  even  during  the  first  year  of  the  war 
were  describing  a  mixture  of  two  or  more  anaerobes  when  they 
thought  they  were  dealing  with  a  pure  culture.  It  was  not  until 
the  development  of  the  newer  anaerobic  methods2  which  made  sur- 
face growths  feasible  that  the  purity  of  anaerobic  cultures  could 
be  relied  upon.  It  is  only  by  repeated  plating  of  these  anaerobic 
spore-bearing  bacilli,  as  emphasized  repeatedly  by  English  workers, 
that  pure  cultures  can  be  obtained.  The  French  workers  have 
adhered  for  the  most  part  to  the  older  method  of  Veillon  which 
consists  of  making  varying  dilutions  of  the  material  to  be  examined 

1  For  the  thorough  revision  of  this  group  of  organisms  we  are  indebted  to  Miss 
Ann  Kuttner,  Instructor  in  this  Laboratory,  Zinsser. 

2  Macintosh  and  Fildes,  Lancet,  1,  1916,  768,  April  8th. 

749 


750  PATHOGENIC   MICROORGANISMS 

in  deep  agar  shake  tubes  until  isolated  colonies  are  obtained.  The 
tube  is  then  filed  through  at  the  level  of  1lie  colony  and  the  isolated 
colony  can  be  fished.  This  is  a  laborious  method  and  is  unreliable. 
Barber3  has  developed  a  technique  whereby  he  can  isolate  a  single 
bacillus  and  has  applied  this  method  to  the  problem  of  the  purifica- 
tion of  the  anaerobic  bacilli. 

The  source  of  anaerobic  bacilli  in  war  wounds  can  always  be 
traced  to  the  contamination  of  the  wound  either  directly  or  in- 
directly with  fecally  contaminated  soil.  Most  of  the  anaerobic 
bacilli  have  been  shown  to  be  normal  inhabitants  of  the  intestinal 
tract  of  man  or  animals,  and  are  present  in  great  numbers  in 
cultivated  ground.  During  the  war  the  incidence  of  anaerobic 
bacilli  in  wounds  and  their  relation  to  the  development  of  gas 
gangrene  was  studied  in  detail. 

In  civilian  practice  gas  gangrene  is  generally  attributed  to  the 
presence  of  B.  Welchii.  In  war  wounds,  however,  it  was  found 
that  although  B.  Welchii  was  isolated  from  the  majority  of  the 
cases  of  gas  gangrene,  it  practically  never  occurred  in  pure  culture, 
and  was  usually  associated,  besides  aerobes,  with  other  anaerobic 
bacilli  which  in  many  instances  proved  to  be  more  pathogenic  than 
B.  Welchii.  The  separation  and  identification  of  these  anaerobic 
bacilli,  and  the  development  of  methods  for  the  control  of  such 
infections  were  among  the  most  important  problems  of  bacteriologists 
during  the  war. 

In  this  book  only  the  most  important  and  frequently  occurring 
anaerobic  bacilli  will  be  discussed.  The  reader  is  referred  for  more 
detailed  description  to  the  book  of  Weinberg  and  Seguin  "La 
Gangrene  Gazeuse,"  and  to  the  Report  of  the  British  Medical 
Research  Committee.  The  anaerobic  bacilli  found  in  war  wounds 
can  be  divided  into  two  general  groups,  as  first  suggested  by  Von 
Hibler4  in  his  book  on  Anaerobes,  published  in  1908 :  the  sacchrolytic 
and  the  proteolytic. 

The  saccharolytic  group  includes  as  its  most  important  members : 
B.  Welchii 
Vibrion  Septique 
B.  Oedematiens 
B.  Fallax 

3  Barber,  Jour.  Exper.  Med.,  32,  1920,  295. 

4  Von  Hibler,  "  Untersuchungen  iiber  die  pathog.  anserob.,"  Jena,  1908. 


THE  ANAEROBIC  BACILLI  751 

The  proteolytic  group  includes: 

B.  sporogcnes 
B.  histolyticus 
B.  putrificus 

This  classification,  like  most  others,  is  not  a  rigid  one.  It  merely 
means  that  members  of  the  saccharolytic  group  have  a  much  greater 
avidity  for  carbohydrates  than  have  members  of  the  proteolytic 
group. 

It  may  be  possible  to  demonstrate  proteolytic  activity  of  the 
organisms  included  in  the  saccharolytic  group  on  special  media  from 
which  all  carbohydrate  has  been  removed,  but  on  the  ordinary 
culture  media  the  difference  between  the  two  groups  is  striking. 

Members  of  the  proteolytic  group  can  be  distinguished  from 
the  saccharolytic  group  by  the  fact  that  they  liquefy  coagulated 
horse  serum.  Organisms  of  the  saccharolytic  group  fail  to  liquefy 
this  medium  even  after  prolonged  incubation.  In  milk  the  saccharo- 
lytic bacilli  produce  acid  and  varying  amounts  of  gas.  The  pro- 
teolytic bacilli  digest  milk  with  the  production  of  alkali. 

The  organisms  of  the  proteolytic  group  are  not  in  themselves 
pathogenic,  but  complicate  wounds  by  their  intense  proteolytic 
action.  They  are  saprophytes,  they  have  no  power  of  invading  the 
tissues  and  if  present  without  members  of  the  saccharolytic  group 
usually  do  not  interfere  with  the  healing  of  the  wound. 

Whether  the  organisms  of  the  saccharolytic  group  are  sapro- 
phytes or  not,  is  an  open  question.  De  Kruif5  concludes  that  B. 
Welchii  cannot  be  classified  as  a  pure  saprophyte,  because  a  twice 
washed  bacillary  emulsion  in  doses  of  0.1  c.c.  to  1  c.c.  will  kill 
a  guinea  pig.  But  in  this  case  even  the  most  careful  intramuscular 
injection  will  kill  some  of  the  tissue  at  the  point  of  inoculation, 
and  with  even  a  very  small  amount  of  dead  tissue,  B.  Welchii  can 
produce  a  toxin  which  has  tremendous  aggressive  action  and  has 
the  property  of  killing  the  tissue  with  which  it  comes  in  contact. 
This  makes  possible  the  further  invasion  of  B.  Welchii. 

B.  Welchii. — B.  Welchii  is  the  organism  most  frequently  found 
in  gas  gangrene.  It  was  present  in  about  72  to  80  per  cent  of  the 
cases  of  gas  gangrene  studied  during  the  war,  and  has  been  con- 
sistently associated  with  civilian  cases  of  gas  gangrene.  It  is  gener- 
ally considered  the  most  important  etiological  factor  in  this  disease. 

*  De  Kruif,  Jour,  Infec.  Dis.,  21,  1917,  No.  6. 


752  PATHOGENIC   MICROORGANISMS 

On  the  other  hand,  it  must  also  be  stated  that  B.  Welchii  was  fre- 
quently present  in  wounds  which  never  developed  gangrene.  Taylor 
reports  that  B.  Welchii  was  found  in  80  per  cent  of  all  wounds 
examined  and  that  only  10  per  cent  of  these  developed  gas  gangrene. 
The  development  of  gas  gangrene  depends  on  the  virulence  of  the 
strain  of  B.  Welchii,  the  amount  of  dead  tissue  present,  and  the 
anaerobic  conditions  in  the  wound.  In  war  wounds  B.  Welchii  was 
practically  never  present  in  pure  culture.  It  was  usually  associated 
with  aerobes  and  with  other  anaerobes  of  the  saccharolytic  and 
the  proteolytic  type. 

B.  Welchii  was  discovered  independently  in  three  countries.  It 
was  first  discovered  by  Welch  and  Nuttall6  in  1892,  and  called  by 
them  Bacillus  aero  genes  capsulatuSj  a  name  still  used  by  the  majority 
of  English  writers.  In  this  country  this  organism  is  usually  called 
B.  Welchii.  Welch  isolated  it  from  the  blood  and  organs  of  a  cadaver 
dead  eight  hours.  In  1893  Fraenkel7  isolated  a  similar  organism  in 
Germany  from  several  cases  of  gaseous  phlegmons,  calling  it  B.  Phleg- 
monis  enephysematosae,  but  soon  recognized  that  he  was  working  with 
the  same  bacillus  previously  described  by  Welch.  However,  this 
organism  is  still  referred  to  as  the  Fraenkel  bacillus  in  German  litera- 
ture. In  1897,  without  having  heard  either  of  Welch's  or  FraenkePs 
work,  this  organism  was  again  described  by  Veilon  and  Zuber8  in 
France  and  called  by  them  B.  perfringens,  B.  Welchii,  Bacillus 
aerogenes  capsulatus,  Fraenkel  bacillus,  B.  perfringens  are  all  names 
for  the  same  organism. 

B.  Welchii  is  a  short,  square  Gram-positive  bacillus,  occurring 
singly  or  in  pairs.  Chains  are  not  formed  as  a  rule.  It  is  non-motile 
and  has  a  capsule.  It  grows  best  under  strictly  anaerobic  condi- 
tions, but  its  requirements  for  anaerobiosis  are  less  rigid  than  those 
of  tetanus.  It  grows  well  in  media  containing  tissue  such  as  cooked 
meat  medium  after  simple  boiling.  With  milk  boiling  is  not  always 
sufficient  to  obtain  good  growth,  and  it  is  best  to  put  milk  tubes 
in  anaerobic  jars.  The  majority  of  strains  do  not  form  spores 
readily.  Alkaline  sugar-free  media  rich  in  protein,  such  as  alkaline 
egg,  are  necessary  to  demonstrate  spore  formation  with  the  majority 
of  strains.  The  spore  of  B.  Welchii  is  large,  oval,  and  central  or 


6  Welch  and  Nuttatt,  Bull.  Johns  Hopkins  Hosp.,  3,  1892,  81. 

7  Fraenkel,  Cent.  f.  Bakt.,  Bd.  13,  1893,  13. 

8  Veillon  and  Zuber,  Arch,  de  Med.  Exper.,  10,  1898,  517. 


THE  ANAEROBIC  BACILLI  753 

subterminal.  B.  Welchii  is  the  most  active  fermenter  in  the  sac- 
charolytic  group.  It  ferments  all  the  common  sugars  with  the  pro- 
duction of  large  amounts  of  gas.  Lactic  and  butyric  are  the  two 
acids  most  frequently  formed,  the  latter  often  giving  cultures  of 
B.  Welchii  a  characteristic  odor.  Glucose  agar  is  sometimes  frag- 
mented to  such  an  extent  that  the  plug  is  blown  off  the  tube. 
Simonds9  has  been  able  to  divide  the  B.  Welchii  group  into  sub- 
divisions depending  on  the  ability  to  ferment  either  glycerine  or 
inulin,  or  both,  or  neither,  and  this  classification  has  been  confirmed 
by  Henry.10  In  wounds,  B.  Welchii  ferments  the  muscle  sugar 
producing  gas  in  the  tissues  and  for  this  reason  is  commonly  called 
the  "gas"  bacillus.  The  crepitation  thus  produced  is  characteristic 
of  gas  gangrene  and  indicates  the  extent  of  the  infection.  The  rapid 
fermentation  of  the  lactose  in  milk  gives  a  characteristic  reaction 
in  this  medium  which  is  diagnostic  for  B.  Welchii.  The  acid  clot 
torn  by  gas  bubbles  and  the  separation  of  the  milk  into  coagulum 
and  whey  is  easily  recognized  and  is  not  given  by  other  anaerobes. 
The  inoculation  of  a  mixed  culture  from  a  wound  into  milk  makes 
possible  the  diagnosis  of  B.  Welchii  within  twelve  to  eighteen  hours. 

Opinions  as  to  the  ability  of  B.  Welchii  to  liquefy  gelatin  vary 
greatly.  B.  Welchii  does  not  grow  well  on  sugar-free  gelatin  and 
it  is,  therefore,  difficult  to  draw  conclusions  as  to  its  action  on 
gelatin.  B.  Welchii  as  pointed  out  by  Rettger  in  1906,11  never 
attacks  proteins  if  carbohydrates  are  present  and  even  in  the  absence 
of  carbohydrate  shows  only  a  very  slight  proteolytic  activity.  No 
indol  is  formed  from  broth,  and  coagulated  serum  is  not  liquefied 
or  blackened. 

The  hemolytic  power  and  pathogenicity  of  different  strains  of 
B.  Welchii  vary  greatly.  B.  Welchii  is  particularly  pathogenic  for 
guinea-pigs  and  pigeons,  the  latter  being  used  in  the  standardization 
of  B.  Welchii  toxin.  B.  Welchii  in  fatal  cases  usually  invades  the 
blood  stream  shortly  before  death,  and  can  usually  be  isolated  from 
the  blood  after  death.  Spores  are  never  formed  in  the  animal  body. 
Rabbits  and  mice  are  much  less  susceptible.  Agglutinin  production 
in  response  to  injections  of  B.  Welchii  in  rabbits  and  horses  is 
extremely  poor.  Simonds  obtained  a  serum  in  rabbits  which  agglu- 
tinated the  homologous  strain  in  a  dilution  of  1—80.  Ten  strains 

9  Simonds,  Mon.  Rock.  Inst.,  No.  5,  1915. 

10  Henry,  Jour.  Pathol.  and  Bacter.,  21,  1917, -344. 

11  Rettger,  Jour.  Biol.  Chem.,  11,  1906,  71. 


754  PATHOGENIC   MICROORGANISMS 

of  B.  Wclchii  failed  to  agglutinate  with  this  scrum,  and  ten  others 
agglutinated  only  in  a  dilution  of  1-20.  The  agglutination  reaction 
for  the  identification  of  anaerobes  has  so  far  proved  unsatisfactory. 
TOXIN  PRODUCTION. — Klose  in  191612  reported  the  isolation  of  a 
toxin  from  B.  Welchii  prepared  by  growing  B.  Welchii  for  fourteen 
days  in  a  5  per  cent  glucose  broth.  The  antitoxin  produced  by 
injections  of  this  toxin  only  protected  guinea-pigs  against  three 
lethal  doses  of  B.  Welchii  cultures.  The  antigenic  properties  of  this 
toxin  were  too  feeble  to  consider  it  a  true  toxin.  The  most  important 
contribution  to  the  bacteriology  of  B.  Welchii  was  made  in  1917 
by  Bull  and  Pritchett13  who  were  able  to  prepare  a  soluble  toxin 
which,  when  injected  into  a  suitable  animal,  produced  a  potent 
antitoxin  possessing  protective  and  curative  properties  against  B. 
Welchii  infections,  in  animals.  One  c.c.  of  antitoxin  per  100  grams 
body  weight  injected  subcutaneously  protected  guinea-pigs  against 
300  lethal  doses  of  culture.14  The  production  of  a  powerful  toxin 
(0.3  c.c.  to  3  c.c.  being  the  M.  L.  D.  for  a  pigeon  of  300  gr.  injected 
intramuscularly)  depended  on  the"  virulence  of  strain,  a  short  in- 
cubation period,  twenty-one  to  twenty-four  hours,  and  the  presence 
of  fresh  muscle  and  glucose  in  the  broth.  Bull  and  Pritchett  found 
no  variations  in  the  ability  of  different  strains  of  B.  Welchii,  irre- 
spective of  the  source  of  the  culture,  to  produce  toxin.  The  toxin 
production  of  less  active  strains  could  be  increased  by  raising  the 
virulence  of  the  culture  by  animal  passage.  Caulfield  in  a  recent 
paper15  finds  that  he  does  not  get  good  toxin  production  unless  the 
virulence  of  his  strain  is  such  that  0.02  c.c.  of  supernatant  fluid 
of  a  young  broth  culture  will  kill  a  300  gr.  pigeon.  Caulfield  also 
emphasizes  the  importance  of  fresh  muscle  in  the  culture  medium, 
although  DeKruif  and  the  Hygienic  Laboratory  in  Washington 
obtained  good  results  by  substituting  chopped  veal  which  can  be 
autoclaved,  for  the  fresh  muscle  tissue.  The  most  potent  toxins, 
however,  seem  to  be  obtained  by  inoculating  the  infected  muscle 
of  a  pigeon  dying  of  a  B.  Welchii  infection  directly  into  the  medium 
to  be  used  for  toxin  production,  or,  at  most,  allowing  one  short 
culture  generation  (ten  hours)  to  intervene  between  the  last  animal 
passage  and  the  inoculation  of  the  broth  for  toxin.  By  preparing 

12  Klose,  Munch,  med.  Woch.,  Bd.  63,  1916,  723. 

13  Bull  and  Pritchett,  Jour.  Exper.  Med.,  26,  1917,  867. 

14  Bull  and  Pritchett,  Jour.  Exper.  Med.,  26,  1917,  867. 
«  Caulfield,  Jour.  Infec.  Dis.,  27,  1920,  No.  2. 


THE  ANAEROBIC  BACILLI  755 

a  toxin  in  this  way,  Bengston  of  Hygienic  Laboratory  has  been  able 
to  prepare  a  B.  Welchii  antitoxin  in  which  1  c.c.  of  serum  contains 
one  unit,  one  unit  neutralizing  1000  M.  L.  D.  of  B.  Welchii  toxin.16 

In  laboratory  animals  infected  with  pure  cultures  of  B.  Welchii, 
the  antitoxin  gives  complete  protection.  Protection  in  laboratory 
animals  is  also  afforded  by  injections  of  antimicrobial  sera  prepared 
by  injections  of  whole  broth  culture  of  B.  Welchii  by  Weinberg  and 
Seguin,  but  the  antitoxin  content  of  these  sera  has  not  been 
determined. 

Bull  and  Pritchett  in  their  original  paper,  state  that  the  toxin 
produced  by  B.  Welchii  is  comparable  to  the  toxins  produced  by 
Tetanus  and  Diphtheria,  and  judged  by  its  antigenic  properties  it 
must  certainly  be  classified  as  a  true  exotoxin.  It  differs  from  the 
classical  toxins  in  that  toxin  production  varies  directly  with  the 
virulence  of  the  strain  and  that  it  has  no  definite  incubation  period. 
B.  Welchii  antitoxin  cannot  protect  against  mixed  infections  where 
B.  Welchii  is  associated  with  either  Vibrion  Septique  or  B.  Oedema- 
tiens.  In  this  case  the  animal  always  dies  of  the  Vibrion  Septique 
or  B.  oedematiens  infection.  However,  since  both  these  organisms 
occur  in  a  smaller  percentage  of  cases  and  have  rarely  been  isolated 
from  civilian  cases  of  gas  gangrene,  B.  Welchii  antitoxin  will  prob- 
ably prove  of  great  value. 

Isolation. — B.  Welchii  is  a  normal  inhabitant  of  the  intestinal 
tract  of  adults  and  may  be  found  in  the  stools  of  infants.  Simonds 
found  B.  Welchii  present  in  eight  out  of  nineteen  stools  of  babies 
under  one  year  of  age.  It  can  be  easily  isolated  from  stools  by 
the  following  procedure:  5  c.c.  of  a  fecal  suspension  in  saline  are 
inoculated  into  a  tube  of  milk  which  has  been  freshly  boiled  and 
cooled.  The  tube  is  heated  at  80°  for  one  hour  to  kill  off  the 
vegetative  forms  of  the  fecal  flora,  and  is  then  incubated.  The 
development  of  the  ''stormy  fermentation"  described  above  indi- 
cates the  presence  of  B.  Welchii.  Purification  is  best  completed 
by  plating  anaerobically  from  the  milk  culture. 

Animal  inoculation  is  also  useful  in  the  isolation  of  B.  Welchii. 
The  material  suspected  of  containing  B.  Welchii  is  injected  intra- 
venously into  a  rabbit.  After  five  minutes  the  rabbit  is  killed  and 
placed  in  the  incubator  for  five  to  eight  hours.  At  the  end  of  this 
time,  the  animal  is  usually  distended  with  gas.  At  autopsy  gas 

16  Bengston,  Hygienic  Laboratory  Bulletin,  No.  122,  1920. 


756  PATHOGENIC   MICROORGANISMS 

bubbles  will  be  found  distributed  throughout  the  organs,  especially 
in  the  liver.  B.  Welchii  if  present,  can  usually  be  isolated  from 
the  liver  and  the  heart's  blood.  Cultures  can  also  be  identified  by 
injecting  them  intramuscularly  into  two  guinea-pigs,  one  normal, 
the  other  protected  by  a  dose  of  B.  Welchii  antitoxin.  If  both 
pigs  die,  and  an  anaerobic  organism  is  isolated  from  the  heart's 
blood,  it  indicates  the  presence  of  some  other  pathogenic  anaerobe, 
not  B.  Welchii.  If  the  normal  pig  dies  with  an  anaerobic,  capsulated, 
non-motile  Gram-positive  bacillus  in  the  heart's  blood,  and  the  anti- 
toxin pig  survives,  it  is  a  fairly  sure  indication  that  the  organism 
in  question  is  B.  Welchii. 

VIBRION  SEPTIQUE  (Synonyms,  Bacillus  of  Oh  on  and  Sachs,  and 
Bacillus  III  of  Von  Hibler). — Vibrion  septique  according  to  Wein- 
berg  and  Seguin,  occurred  in  12  per  cent  of  the  wounds  examined 
by  them.  Henry  isolated  it  in  16  per  cent  of  his  cases.  Before  the 
war,  cases  of  human  gas  gangrene  due  to  vibrion  septique  alone 
were  very  few  in  number.  Such  cases  were  described  by  Ghon 
and  Sachs,17  by  Von  Hibler,  Gould,18  and  by  Muir  and  Ritchie.19 
During  the  war,  vibrion  septique  was  usually  associated  with  other 
anaerobes,  notably  B.  Welchii.  Weinberg  and  Seguin  cite  only  one 
case  of  gas  gangrene  in  which  vibrion  septique  was  the  only  anaerobe 
present. 

Vibrion  septique  was  first  described  by  Pasteur20  in  1877  who 
isolated  it  from  the  blood  of  a  cow  dead  three  days,  and  from  the 
blood  of  a  horse  dead  one  day,  both  animals  having  supposedly  died 
of  anthrax.  Pasteur  called  this  organism  a  vibrion,  although  it  is 
in  reality  a  bacillus,  because  it  is  extremely  motile  in  animal  exudates 
and  may  look  slightly  curved  when  in  motion.  In  1881,  Koch21  in 
studying  the  etiology  of  anthrax,  isolated  an  organism  which  he 
called  the  bacillus  of  malignant  edema.  Koch  considered  his 
organism  identical  with  Pasteur's  vibrion  septique,  although  the 
bacillus  of  malignant  edema  had  marked  proteolytic  properties, 
which  Pasteur  did  not  mention  in  the  description  of  vibrion  septique. 
A  great  amount  of  confusion  has  arisen  out  of  this  controversy 
and  the  literature  is  full  of  papers  discussing  whether  or  not  Pasteur 

17  Ghon  and  Sachs,  Cent,  f .  Bakt.,  1  Abt.  Orig.,  48,  1909,  396. 

18  Gould.,  Annals  of  Surgery,  38,  1903,  481. 

19  Muir  and  Ritchie,  Manual  of  Bacteriology,  2d  Edit.,  Edinburgh,  1899. 

20  Pasteur  et  Jourbet,  Bull,  de  1'Acad.  Med.,  Scien.,  Vol.  6,  793. 

21  Koch,  Mitt.  a.  d.  k.  Gesundheitsamt,,  1,  1881,  53*. 


THE  ANAEROBIC  BACILLI  757 

and  Koch  were  working  with  the  same  organisms  and  with  attempts 
to  identify  organisms  isolated  from  wounds  with  one  or  the  other 
of  these  bacilli.  The  majority  of  workers  now  consider  that  Pasteur 
was  working  with  a  strictly  saccharolytic  organism  which  is  identical 
with  what  we  call  vibrion  septique  at  the  present  time.  The  bacillus 
of  malignant  edema  of  Koch  is  thought  by  most  investigators  to 
belong  to  the  proteolytic  group  and  is  fairly  definitely  identified  with 
B.  sporo  genes. 

Vibrion  septique  is  a  motile,  slender  Gram-positive  bacillus  with 
slightly  rounded  ends.  It  is  a  strict  anaerobe.  It  forms  spores 
readily  in  most  media.  The  spore  is  oval,  occurring  either  centrally 
or  subterminally,  and  appears  at  the  end  of  twenty-four  to  forty- 
eight  hours.  It  has  no  capsule.  It  ferments  the  common  sugars 
with  the  exception  of  saccharose.  It  produces  a  loose  clot  in  milk 
in  one  to  four  days.  Gelatin  is  liquefied,  but  coagulated  serum  is 
not  attacked.  Vibrion  septique  is  hemolytic.  It  is  always  pathogenic 
for  laboratory  animals  and  guinea-pigs,  mice,  pigeons  and  rabbits 
are  all  susceptible.  It  invades  the  blood  stream  producing  a  sep- 
ticemia.  The  occurrence  of  long  filamentous  forms  in  the  livers  of 
guinea-pigs  dying  of  a  vibrion  septique  infection  is  characteristic 
and  is  used  in  the  identification  of  vibrion  septique. 

Robertson22  has  recently  divided  the  vibrion  septique  group  into 
four  serological  types,  based  upon  the  agglutination  reaction.  Miss 
Robertson  again  stresses  the  necessity  of  minute  care  in  purifying 
cultures  and  points  out  that  impure  cultures  fail  to  agglutinate. 
It  is  difficult,  however,  to  attach  much  importance  to  variations  in 
agglutinin  production  of  different  strains,  since  there  is  no  differ- 
ence in  toxin  production,  and  since  the  antitoxin  produced  by  the 
injection  of  the  toxin  of  strains  belonging  to  one  serological  type 
neutralizes  the  toxins  produced  by  members  of  the  other  types. 
The  agglutination  reaction  in  the  case  of  vibrion  septique  subdivides 
strains  that  agree  in  every  other  respect,  and  may  in  this  instance 
be  regarded  as  ultraspecific,  as  Miss  Robertson  suggests.  She  was 
able  to  obtain  agglutinating  sera  with  a  titer  of  1-25,000. 

TOXIN  PRODUCTION. — A  powerful  soluble  toxin  is  produced  by  all 
strains  of  vibrion  septique  and  does  not  depend  on  the  virulence 
of  the  culture.  According  to  Robertson  as  potent  toxins  are 
produced  by  old  laboratory  strains  of  vibrion  septique  as  by  recently 

22  Robertson,  Jour.  Bacter.  and  Pathol.,  1920, 


758  PATHOGENIC   MICROORGANISMS 

isolated  cultures.  The  toxin,  like  that  of  B.  Welehii,  has  no  incuba- 
tion period.  The  toxin  of  vibrion  septique  often  fails  to  produce 
death  in  guinea  pigs  when  injected  subcutaneously  or  intramus- 
cularly, merely  producing  local  necrosis.  Toxin  production  is  tested 
both  in  rabbits  and  guinea  pigs  by  intravenous  injection.  0.5  c.c. 
of  toxin  injected  intravenously  kills  a  guinea  pig  in  five  minutes. 
0.1  to  1  c.c.  injected  into  rabbits  intravenously  kills  them  without 
a  latent  period,  with  symptoms  of  respiratory  disturbance,  paralysis 
and  convulsions.  It  is  difficult  to  establish  an  M.  L.  D.  for  rabbits 
of  the  same  v/eight  owing  to  individual  variation.  In  some  instances, 
death  is  produced  immediately  in  one  rabbit,  whereas  another  rabbit 
of  the  same  weight  will  show  severe  symptoms  followed  by  recovery. 

METHOD  OF  PRODUCING  TOXIN. — The  Hygienic  Laboratory  obtains 
a  powerful  toxin  using  a  0.2  per  cent  glucose  broth  containing  10 
per  cent  horse  serum.  Robertson  recommends  using  liver  of  a  pig 
dying  of  a  vibrion  septique  infection  with  which  to  inoculate  the  broth, 
but  the  difficulties  of  obtaining  a  liver  without  gross  contaminations 
are  such  that  the  former  method  is  preferable.  The  broth  is  in- 
cubated twenty-four  to  forty-eight  hours.  Care  should  be  taken 
in  the  selection  of  the  filter  since  certain  filters  seem  to  hold  back 
a  large  percentage  of  the  toxin.  This  point  is  emphasized  both  by 
Weinberg  and  Seguin  and  by  Robertson.  Antitoxins  are  prepared 
by  injecting  the  toxin  into  horses  or  sheep.  The  French  standard 
requires  that  1/1000  c.c.  of  the  antitoxin  should  neutralize  two  fatal 
doses  of  the  toxin  after  thirty  minutes  incubation  of  the  mixture 
at  room  temperature.  The  vibrion  septique  antitoxin  is  specific; 
it  does  not  protect  against  B.  oedematiens. 

OCCURRENCE. — Vibrion  septique  has  been  isolated  from  milk. 
Heller23  in  an  excellent  summary  of  anaerobic  infections  in  animals 
has  shown  that  spontaneous  infections  by  vibrion  septique  occur 
in  sheep,  horses,  and  hogs.  Meyer24  in  1915  reported  the  isolation 
of  typical  vibrion  septique  from  two  cases  of  symptomatic  anthrax 
in  hogs.  Cattle,  according  to  Heller,  are  less  susceptible  to  vibrion 
septique  infections  than  the  other  animals  mentioned.  Herbivorous 
animals  are  subject  to  infections  with  vibrion  septique,  both  follow- 
lowing  and  not  following  demonstrable  wounds,  whereas  infections 
in  man  seem  to  occur  only  as  the  result  of  wounds. 


"  Heller,  Jour.  Infec.  Dis.,  27,  1920,  385. 
24  Meyer,  Jour.  Infec.  Dis.,  12,  1915,  458. 


THE  ANAEROBIC  BACILLI  759 

Differentiation  of  Vibrion  Septique  and  B.  Chauvaei  (Bacillus 
of  symptomatic  anthrax,  Blackleg). — B.  chauvaei  was  in  no  instance 
isolated  from  wound  cultures,  and  has  never  been  known  to  cause 
an  infection  in  man.  Vibrion  septique,  on  the  other  hand,  frequently 
infects  animals  and  a  bacteriological  differentiation  between  vibrion 
septique  and  B.  chauvaei  must  be  made.  These  two  organisms  are 
closely  related  and  very  similar,  and  a  reliable  differentiation  is 
difficult  even  for  a  bacteriologist  familiar  with  anaerobic  bacilli. 
Robertson  distinguishes  between  B.  chauvaei  and  vibrion  septique 
by  the  fact  that  the  former  ferment  saccharose  and  not  salicin, 
whereas  vibrion  septique  ferments  salicin  and  not  saccharose.  Long 
snake-like  filaments  are  demonstrable  in  smears  from  the  liver  of 
guinea-pigs  dead  of  vibrion  septique  infection;  these  are  entirely 
lacking  in  B.  chauvaei  infections.  Vibrion  septique  is  more  patho- 
genic for  laboratory  animals  and  produces  more  gas  in  the  tissues 
than  B.  chauvaei.  B.  chauvaei  grows  more  slowly  than  vibrion 
septique.  Vibrion  septique  is  Gram-positive,  whereas  most  inves- 
tigators consider  B.  chauvaei  Gram-negative.  Protection  tests  with 
a  known  vibrion  'septique  antitoxin  ought  to  prove  the  most  reliable 
way  of  identifying  vibrion  septique. 

B.  oedematiens. — Weinberg  and  Seguin  claim  to  have  isolated 
this  organism  in  34  per  cent  of  the  wounds  examined  by  them.  This 
is  a  higher  proportion  than  that  obtained  by  other  workers.  Henry 
found  B.  oedematiens  in  five  out  of  fifty  cases  examined.  B.  oedema- 
tiens was  isolated  in  1915  by  Weinberg  and  Seguin.  In  1916  a 
similar  organism  was  isolated  by  Sacquepee25  under  the  name 
"bacille  de  Toedeme  gazeuse  malm."  Later  Sacquepee  called  this 
organism  B.  Bellonensis.  B.  Bellonensis  and  B.  oedematiens  are 
now  considered  to  be  the  same  organism  by  the  majority  of  workers. 
According  to  Heller,  B.  oedematiens  is  closely  related  to  but  not 
identical  with  a  bacillus  discovered  in  1894  by  Novy  and  called 
by  him  B.  oedematiens  maligin  II.26  B.  oedematiens  is  a  strict 
anaerobe.  It  is  a  large  Gram-positive  bacillus,  resembling  anthrax 
in  appearance.  It  is  practically  non-motile.  It  forms  chains  in 
culture  and  often  shows  curved  forms  after  two  or  three  days. 
Filaments  are  not  formed  in  the  animal  body.  It  forms  oval  sub- 
terminal  spores  readily  in  all  media.  It  ferments  most  of  the 


26  Sacquepee,  Ann.  de  1'inst.  Past.,  30,  1916,  76. 
,  Zeit.  f.  Hyg.,  17,  1894,  209. 


760  PATHOGENIC   MICROORGANISMS 

common  sugars,27  and  forms  a  loose  clot  in  milk  in  three  or  four 
days.  It  liquefies  gelatin,  but  does  not  attack  coagulated  serum. 

PATHOGENICITY. — B.  oedematiens  is  usually  pathogenic,  although 
Weinberg  and  Seguin  report  the  isolation  of  two  non-virulent  strains, 
rabbits  are  less  susceptible  than  guinea-pigs.  Mice  are  also  sus- 
ceptible. It  may  or  may  not  enter  the  blood  stream.  The  lesion 
in  the  animal  is  characterized  by  a  whitish  gelatinous  exudate  and 
the  absence  of  gas.  The*  production  of  agglutinating  sera  with  B. 
oedematiens  has  not  been  satisfactory,  because  B.  oedematiens  tends 
to  agglutinate  spontaneously.  It  is  feebly  hemolytic  much  less  so 
than  vibrion  septique  and  B.  perfringens. 

TOXIN. — B.  oedematiens  forms  a  soluble  toxin.  Different  strains 
vary  in  their  toxin  production.  With  a  potent  strain  1/100  c.c.  of 
toxin  injected  intravenously  kills  a  300-400-gram  guinea-pig  in 
forty-eight  hours.  This  toxin  differs  from  those  of  vibrion  septique 
and  B.  Welchii  in  that  it  never  kills  acutely  on  intravenous  injection. 
The  toxin  is  prepared  according  to  Weinberg  and  Seguin,  by  grow- 
ing B.  oedematiens  in  broth  containing  chopped  veal  for  six  to  ten 
days. 

ANTITOXIN. — Rabbits,  sheep  and  horses  have  been  used  to  produce 
antitoxic  sera.  Immunization  is  difficult  and  small  doses  must  be 
used  at  first.  Weinberg  and  Seguin  prepared  an  antitoxin  in  a  horse 
of  such  a  titer  that  1/10,000  dilution  neutralized  two  lethal  doses 
(guinea-pig). 

B.  Fallax. — B.  fallax  was  discovered  during  the  war  by  Wein- 
berg and  Seguin.  It  is  a  much  less  important  factor  in  gas  gangrene 
than  the  members  of  the  saccharolytic  group  already  described.  It 
is. usually  associated  with  other  pathogenic  anaerobes.  Weinberg 
and  Seguin  cite  one  case  in  which  it  invaded  the  blood  stream  and 
caused  death.  It  was  isolated  by  Henry  in  three  cases  out  of  a  series 
of  fifty. 

It  is  an  anaerobic  Gram-positive  bacillus,  resembling  vibrion 
septique  in  appearance.  It  has  a  capsule  and  is  slightly  motile.  It 
does  not  form  spores  readily  in  most  culture  media.  Spores  are 
formed  on  coagulated  serum  and  are  subterminal. 

B.  fallax  coagulates  milk  very  slowly.  It  does  not  liquefy  gelatin 
or  coagulated  serum. 

B.  fallax  is  not  hemolytic.  It  is  only  slightly  pathogenic  and 
soon  becomes  avirulent  on  artificial  cultivation. 

27  Wolf,  Jour.  Pathol.  and  Bacter.,  23,  1920,  254. 


THE  ANAREOBIC  BACILLI  761 

Proteolytic  Group. — The  organisms  of  this  group  can  never 
produce  gas  gangrene  without  the  presence  of  one  or  more  bacilli 
of  the  saccharolytic  group.  The  members  of  the  proteolytic  group 
digest  milk  without  the  formation  of  a  clot  and  liquefy  and  often 
blacken  coagulated  serum.  These  two  characteristics  together  with 
the  fact  that  cultures  of  the  proteolytic  organisms  usually  have  a 
very  offensive  odor,  make  it  comparatively  easy  to  distinguish  them 
from  the  saccharolytic  group.  Sugars  are  fermented  by  the  pro- 
teolytic type,  but  much  less  rapidly  and  with  the  production  of 
less  acid  and  gas  than  in  the  case  of  the  saccharolytic  group.  The 
members  of  the  proteolytic  group  produce  spores  readily  in  all 
media.  None  of  these  organisms  are  very  pathogenic  and  produce 
no  general  picture  of  toxemia  in  spite  of  the  tremendous  liquefaction 
of  tissue  caused  by  them.  The  ferments28  of  several  of  the  pro- 
teolytic anaerobes  have  been  isolated  and  have  been  found  to  split 
proteins  to  animo  acids  so  rapidly  that  there  is  no  time  for  the 
intermediate  products  to  intoxicate  the  animals.  The  separation 
of  the  proteolytic  anaerobes  from  the  saccharolytic  is  extremely 
difficult.  The  members  of  the  two  groups  are  usually  present  to- 
gether, arid  what  will  seern  to  be  a  pure  culture  of  a  saccharolytic 
organism  if  held  for  any  length  of  time,  will  often  show  a  con- 
tamination with  a  proteolytic  organism.  The  best  methods  for 
separation  of  the  two  groups  are :  by  rapid  transplantation  in  sugar 
media,  where  the  saccharolytic  organisms  outgrow  the  proteolytic, 
combined  with  frequent  plating,  or  by  animal  inoculation.  The 
latter  is  the  most  satisfactory.  In  the  animal  body  after  intramus- 
cular injection,  the  more  pathogenic  organisms  belonging  to  the 
saccharolytic  group  frequently  invade  the  blood  stream  and  may 
be  isolated  from  the  heart's  blood. 

B.  sporogenes. — This  organism  was  next  to  B.  Welchii  most 
frequently  found  in  wound  cultures.  Weinberg  and  Seguin  isolated 
it  in  27  per  cent  of  their  cases.  B.  sporogenes  was  the  anaerobe 
usually  responsible  for  the  foul  odor  of  wounds.  According  to  most 
authors,  the  pathogenicity  of  this  organism  is  negligible.  Weinberg 
and  Seguin  claim  to  have  isolated  a  few  toxic  strains,  but  these 
may  possibly  have  been  mixed  with  members  of  the  saccharolytic 
group.  Heller  has  not  found  any  proteolytic  anaerobes  that  are 
pathogenic  for  animals. 

23  Blanc  and  Pozerski,  Compt.  Rend.  Soc.  Biol.,  87,  1920,  29. 


762  PATHOGENIC   MICROORGANISMS 

B.  sporogencs  was  definitely  described  by  Metchnikoft'29  in  1908. 
Whether  B.  sporogenes  is  identical  or  not  with  Koch's  bacillus  of 
malignant  edema,  will  probably  never  be  definitely  settled.  It  is 
considered  identical  by  many  workers,  although  this  is  emphatically 
denied  by  others.  B.  sporogenes  is  a  Gram-positive,  anaerobic 
bacillus,  actively  motile,  forming  oval  subterminal  spores  readily 
in  all  media  and  in  the  animal  body.  It  is  intensely  proteolytic, 
liquefying  gelatin  and  coagulated  serum,  and  digesting  and  blacken- 
ing meat.  Most  strains  of  B.  sporogenes  are  not  hemolytic.  Occa- 
sionally a  feebly  hemolytic  strain  has  been  isolated.  It  does  not 
produce  a  soluble  toxin  and  is  not  pathogenic  for  laboratory  animals 
unless  injected  in  large  quantities. 

B.  Histolyticus. — This  organism  was  discovered  by  Weinberg 
and  Seguin  and  isolated  by  them  from  eight  wound  cultures.  Like 
B.  sporogenes,  it  is  intensely  proteolytic  and  is  of  interest  chiefly 
because  of  the  striking  lesion  it  produces  in  the  animal  body.  It 
is  a  Gram-positive  anaerobic,  motile  bacillus  with  rounded  ends. 
Sporulation  takes  place  in  all  media,  different  strains  varying  in 
the  time  required  for  spore  formation.  The  spores  are  large  and 
oval,  and  occupy  a  terminal  position.  No  gas  is  formed  in  cultures 
of  B.  histolyticus  and  no  putrid  odor  develops.  Gelatin  and  coagu- 
lated serum  are  liquefied.  It  does  not  produce  a  soluble  toxin.  It 
is  not  hemolytic.  The  injection  of  large  doses,  2  to  3  c.c.  intra- 
muscularly into  guinea  pigs,  of  the  whole  culture  digests  the  tissues 
so  rapidly  that  at  the  end  of  twelve  to  twenty-four  hours,  the  bone 
may  be  exposed.  The  picture  is  striking,  one  of  the  characteristics 
of  B.  histolytic  infection  being  that  in  spite  of  a  tremendous  local 
lesion,  the  animal  appears  well. 

B.  putrificus. — B.  putrificus  was  occasionally  found  in  putrid 
wounds.  It  was  first  discovered  in  1884  by  Bienstock  and  is  char- 
acterized by  a  terminal  oval  spore.  Bienstock  isolated  B.  putrificus 
from  the  intestine  of  a  cadaver.  It  is  a  Gram-positive  anaerobe,  motile, 
forming  spores  in  all  media.  It  is  actively  proteolytic,  producing 
a  foul  odor.  No  pathogenic  strains  have  been  isolated.  It  has  been 
studied  by  Tissier  and  Martally  who  found  it  in  putrid  meat,  Klein 
worked  with  a  similar  organism  which  he  called  B.  sporogenes 
cadaveris.  Hibler  considers  B.  putrificus  and  B.  sporogenes  cada- 
veris  the  same. 


29  Metchnikoff,  Ann.  de  1'Inst.  Past,,  22,  1908,  419. 


THE  ANAEROBIC  BACILLI  763 

Identification  of  Anaerobes  Present  in  Wound  Cultures. — From 
the  point  of  serum  treatment  of  infected  wounds,  the  prompt  iden- 
tification of  the  members  of  saccharolytic  group,  B.  Welchii,  B. 
oedematiens  and  Vibrion  Septique,  is  most  important.  The  process 
of  purification  and  identification  by  cultural  methods  is  at  best  slow, 
and  Henry30  has,  therefore,  suggested  the  inoculation  of  the  unknown 
material  into  immunized  guinea-pigs  as  the  quickest  and  most 
reliable  method.  The  procedure  he  outlines  is  as  follows :  inoculate 
the  unknown  mixed  culture  into  cooked  meat  medium,  and  incubate. 
The  next  day  inoculate  the  supernatant  fluid  into  milk,  and  inject  in- 
tramuscularly into  two  immunized  guinea-pigs,  one  pig  having  received 
a  mixture  of  B.  Welchii  and  Vibrion  Septique  antitoxin,  the  other  a 
mixture  of  B.  Welchii  and  B.  oedematiens.  The  stormy  fermenta- 
tion of  milk  is  diagnostic  for  B.  Welchii  and  this  reaction  takes  place 
within  twenty-four  hours.  If  the  pig  that  was  protected  against 
Vibrion  Septique  (the  B.  Welchii  factor  having  been  eliminated  in 
both  pigs)  dies,  it  indicates  the  presence  of  some  other  pathogenic 
anaerobe,  probably  B.  oedematiens.  The  diagnosis  of  B.  oedematiens 
is  further  indicated  if  the  guinea-pig  that  received  the  B.  oedematiens 
combination  of  sera  survives.  If  the  animal  inoculations  come  out 
in  the  opposite  way,  the  presence  of  Vibrion  Septique  is  indicated. 
The  pathogenic  organism  can  usually  be  isolated  from  the  heart's 
blood  of  the  animal  that  succumbs.  By  using  a  "filter"  of  protected 
guinea-pigs  in  this  way,  the  pathogenic  organisms  can  be  separated 
out  and  the  specific  sferum  injected  into  the  patient  within  forty-eight 
hours. 

THE  COOPERATION  OF  SURGERY  AND  BACTERIOLOGY  IN 
THE  MANAGEMENT  OF  TRAUMATIC  WOUNDS  (WAR 
WOUNDS) 

The  extensive  experience  gained  by  surgeons,  during  the  war, 
in  connection  with  infected  wounds  has  developed  a  number  of 
important  bacteriological  methods  which  are  likely  to  remain  as 
parts  of  the  routine  work  of  civil  hospitals,  especially  those  in 
which  traumatic  cases  are  handled. 

It  is  not  our  intention  to  go  into  the  various  problems  and  con- 
troversies that  have  arisen  among  surgeons  concerning  the  value 
of  irrigation  with  Dakin's  solution  or  with  other  antiseptics.  This 

30  Henry  and  Lacy,  Jour.  Pathol.  and  Bacter.,  32,  1920,  No.  3. 


764  PATHOGENIC   MICROORGANISMS 

is  not  directly  concerned  with  the  question  of  bacteriological  control, 
a  matter  which  is  desirable  and  seems  eminently  logical  whatever 
method  the  surgeon  chooses  to  use  for  the  disinfection  of  the  wound. 
It  is,  however,  chiefly  in  connection  with  Dakin's  solution  irrigation 
that  this  method  was  developed  by  Carrel.  The  most  complete 
treatise  on  the  entire  matter  may  be  found  in  the  book  lay  Carrel 
and  De  Helly,  "The  Treatment  of  Infected  Wounds,"  (Hocber, 
New  York,  1919). 

The  usual  type  of  war  wound  or,  for  that  matter,  any  kind  of 
traumatic  wound,  presents  conditions  in  regard  to  the  possibilities 
of  infection  which  are  quite  different  from  those  ordinarily  en- 
countered in  aseptic  surgery.  From  the  skin  and  clothing  bacteria, 
both  aerobic  and  anaerobic,  are  carried  by  the  projectiles  or  other 
foreign  bodies  into  the  tissues.  Tissues  are  destroyed  to  a  variable 
degree,  and  such  devitalized  tissues  furnish  an  excellent  medium 
for  bacterial  growth.  There  is  always  an  interval  or  latent  period 
between  contamination  of  the  wound  and  proliferation  and  penetra- 
tion of  the  organisms.  The  duration  of  this  latent  period  varies, 
but  usually  approximates  six  hours. 

The  immediate  aim  of  treatment  is  the  prevention  or  limitation 
of  infection,  and,  for  this  reason,  the  rational  method  of  determining 
whether  this  purpose  is  being  accomplished  and  what  the  next 
procedure  should  be  is  bacteriological  control. 

The  first  step  in  limiting  infection  in  such  wounds  is  accom- 
plished by  debridement,  that  is,  excision  of  the  tract  with  removal 
of  all  the  devitalized  and  contaminated  tissues,  together  with  foreign 
bodies,  bits  of  projectile,  clothing,  etc.  Bacteria  are  greatly 
diminished  though  not  eradicated  by  this  procedure. 

Bacteriological  control  of  the  original  infection  of  the  wound  and 
its  progress  under  treatment  is  carried  out  by  a  method  of  systematic 
smear  examination  of  the  wound,  supplemented  by  cultures,  first 
practically  developed  by  Carrel. 

The  smear  method,  introduced  by  Carrel  and  employed  since  that 
time  by  many  surgeons  on  a  large  material,  is  simple,  can  be  carried 
out  by  any  well  trained  assistant  or  technician  without  the  aid  of  a 
highly  trained  bacteriologist,  and  has  apparently  yielded  results  of 
value.  Our  description  is  taken  almost  entirely  from  Carrel 's  own  writ- 
ings. "Wounds  should  be  examined  every  two  or  three  days,  and  when 
the  time  for  secondary  closure  appears,  perhaps  every  day.  The 
principle  consists  in  the  examination  of  the  secretions  of  the  wounds 


THE  ANAEROBIC  BACILLI  765 

by  means  of  smears  in  such  a  way  that  an  approximate  estimate 
of  the  number  of  bacteria  contained  in  the  wounds  can  be  made. 
Although  the  method  is  very  inaccurate,  its  value  does  not  depend 
upon  its  revealing  slight  differences,  the  significant  variations  being 
so  widely  apart  that  the  necessary  error  in  the  comparative  enumera- 
tions does  not  render  the  method  useless. 

The  examination  need  not  begin  earlier  than  twelve  hours  after 
the  infliction  of  the  wound,  since  up  to  that  time  few  bacteria  will 
be  found.  At  the  end  of  this  time,  when  hemorrhage  has  completely 
stopped,  smears  are  taken  with  a  platinum  loop  from  different  parts 
of  the  wound.  The  points  from  which  cultures  are  taken  should 
always  be  those  in  which  bacteria  are  most  likely  to  be  present 
in  large  numbers.  Thus,  Carrel  chooses  points  in  contact  with 
foreign  bodies,  necrotic  bits  of  bone,  and  from  deep  in  the  sinuses 
and  crevices  of  the  wound.  Specimens  should  never  be  taken  from 
bleeding  points.  Specimens  should  always  be  taken  from  a  consider- 
able number  of  different  places  in  the  same  wound.  Care  should  be 
exercised  to  avoid  taking  smears  from  the  skin  adjacent  to  the 
wound.  With  the  end  of  a  small  platinum  loop  small  amounts  of 
secretion  are  picked  up,  and  smeared  upon  slides  in  such  a  way 
that  approximately  the  same  area  is  covered  by  the  different 
loopfuls  of  secretion.  With  loops  of  uniform  size  and  a  little  prac- 
tice, a  surprising  uniformity  of  technique  can  be  developed. 

These  smears  are  allowed  to  dry,  and  may  be  stained  in  a 
variety  of  ways.  Carbol  thionin  has  been  extensively  used,  but  we 
believe  that  a  Gram  stain  which  is  almost  as  simple,  will  give  a 
little  more  useful  information. 

The  stained  slides  can  now  be  examined  under  the  microscope 
and  the  number  of  bacteria  per  field,  counted.  If  the  number  exceeds 
fifty  or  more  to  the  field,  more  accurate  counting  will  yield  no 
valuable  information  because  the  wound  still  contains  too  many 
bacteria  to  warrant  closure  or  relaxation  of  the  local  therapy  that 
is  being  applied. 

Gradually,  as  the  wound  improves,  less  and  less  bacteria  will 
appear  in  the  daily  series  of  slides,  and  when  it  has  dropped  below 
fifty  per  field,  careful  counting  may  give  an  index  of  daily  variations. 
Eventually,  they  will  drop  to  only  one  microorganism  per  five,  ten 
or  twenty  fields,  in  which  case  the  daily  report  can  be  expressed  in 
fractions,  as  1/5,  1/10,  or  1/20,  etc.  The  daily  counts  can,  in  this 
way,  be  numerically  charted,  and  constructed  into  a  curve  which 


766  PATHOGENIC   MICROORGANISMS 

will  show  the  surgeon  by  a  giance*  the  numerical  progress  of  the 
baeterial  infection. 

Carrel  states  that  it  is  useless  to  take  any  smears  as  long  as 
hemorrhage  exists.  If  the  wound  is  being  irrigated  with  Dakin's 
solution  or  other  antiseptic  fluids,  the  treatment  must  be  omitted 
for  at  least  two  hours  before  the  smears  are  taken.  Smears  taken 
from  the  surface  of  smooth  muscles  are  practically  useless,  since 
smooth  muscle  becomes  sterile  early  in  the  healing  process.  There- 
fore, the  choosing  of  the  point  of  smear  is  of  the  utmost  importance. 
The  depth  of  the  wound  may  begin  to  become  sterile  at  times  when 
individual  little  foci  around  necrotic  bone,  small  pockets,  etc.,  may 
still  contain  numerous  bacteria.  This  must  be  borne  in  mind  and 
an  intelligent  survey  of  the  wound  made  by  the  bacteriologist  who 
takes  the  smear.  To  overlook  such  dangerous  points  would  seriously 
imperil  the  life  of  the  patient,  were  the  wound  closed.  When 
absolutely  no  bacteria  are  found  in  such  smears,  it  does  not  mean 
that  the  wound  is  completely  sterile.  It  is  still  possible  that  cultures 
might  reveal  organisms,  and  when  the  period  of  secondary  closure 
approaches,  especially  when  streptococci  have  been  present  at  a 
previous  time,  we  would  regard  it  of  the  greatest  importance  to 
take  a  culture  aimed  particularly  at  the  demonstration  of  hemolytic 
streptococci,  before  the  actual  suture  is  carried  out. 

Cultural  examinations  should  be  made  at  the  beginning  by  taking 
specimens  from  parts  of  the  wound  selected  as  indicated  above, 
and  smearing  them  upon  fresh  blood-agar  plates  (without  glucose). 
This  is  primarily  aimed  at  determining  whether  cocci,  and  especially 
hemolytic  streptococci  or  staphylococci,  are  present.  If  the  smears 
show  a  great  many  bacilli  resembling  the  ordinary  anaerobes,  it 
may  be  well,  too,  to  make  anaerobic  cultures,  but  anaerobic  analysis 
is  not  of  great  immediate  value  to  the  surgeon  as  far  as  further 
procedure  is  concerned  because  of  the  long  time  consumed  by  such 
examinations.  Suture  is  not  carried  out  if  hemolytic  cocci  of  any 
kind  are  present,  and  for  this  season,  with  a  smear  as  a  preliminary 
indication,  frequent  culture  upon  blood  plates  should  be  undertaken 
during  the  progress  of  the  treatment. 

In  discussing  the  subject,  it  is  not  possible  to  give  an  intelligent 
survey  of  the  bacteriological  methods,  without,  to  some  extent, 
entering  into  the  surgical  considerations  involved.  For  this  reason 
.we  quote  from  Pool,31  whose  experience  with  this  type  of  wound 
has  been  extensive. 

«  Pool,  E.  H.,  Jour,  A.  M,  A.,  73,  1919. 


THE  ANAEROBIC  BACILLI  767 

Debridement  should  be  carried  out  as  soon  as  possible  after 
infliction  of  the  wound.  Primary  suture  may  be  employed  only 
during  quiet  periods  in  case  of  war,  and  in  hospitals  where  the 
patient  may  be  retained  for  careful  observation.  Otherwise  suture 
of  the  wound  may  lead  to  enclosure,  within  an  imperfectly  debrided 
wound,  of  various  microorganisms,  including  those  which  produce 
gas  gangrene.  In  regard  to  delayed  primary  and  secondary  suture, 
the  following  observations  of  Dr.  E.  H.  Pool  are  not  without  interest. 

"The  determination  as  to  when  a  wound  may  be  sutured  depends  on  bac- 
teriologic  findings  and  clinical  observation.  It  must  be  emphasized  that  the 
co-operation  of  a  bacteriologist  is  indispensable  in  making  a  decision  aS  to  the 
indications  for  delayed  primary  and  secondary  sutures.  The  practical  func- 
tion and  indisputable  importance  of  the  bacteriologist  in  war  surgery  lies  in 
this.  In  the  consideration  as  to  whether  a  wound  is  suturable  or  not,  reliance 
must  be  placed  chiefly  on  cultures,  the  important  feature  being  the  determina- 
tion of  the  presence  or  absence  of  hemolytic  cocci.  For  this,  a  routine  blood- 
agar  examination  is  essential. 

Bacterial  counts  are  far  from  exact,  yet  they  give  an  in.dication  as  to  the 
degree  of  bacterial  contamination  of  a  wound,  especially  the  progress  from 
day  to  day,  and  are  of  value  especially  for  one  untrained  in  estimating 
clinically  the  indications  and  contraindications  for  suture. 

From  eighteen  to  forty-eight  hours  after  the  original  operation  of  de- 
bridement  or  excision  of  tissues,  the  wound  is  dressed  and  a  culture  and  a 
smear  are  made.  A  report  is  returned  as  soon  as  possible.  This  contains  the 
approximate  number  of  organisms  per  field  and  the  varieties  of  organisms. 
If  no  organisms  are  found,  suture  is  indicated.  If  hemolytic  cocci  are  present, 
suture  is  not  considered.  In  the  absence  of  hemolytic  cocci,  if  the  wound  is 
clinically  suturable,  the  presence  of  a  few  anaerobes  or  other  organisms 
(approximately  one  in  two  fields)  does  not  contraindicate  suture.  A  con- 
siderable number  of  organisms  of  any  kind  indicates  delay  of  suture,  until 
the  bacterial  growth  declines.  A  culture  and  a  smear  should  be  repeated  at 
the  following  dressing ;  the  results  of  this  examination  will  determine  suturing 
or  further  delay.  If  the  wound  is  left  open  for  a  considerable  period,  e.g., 
over  a  week,  or  is  definitely  infected,  a  smear  is  made  every  two  days.  It  is 
also  advisable  to  make  a  culture  occasionally.  Care  must  be  taken  not  to 
touch  the  skin  surface  in  making  the  smear,  since  skin  contamination  vitiates 
the  value  of  the  report.  From  the  smear  a  bacterial  curve  is  plotted  accord- 
ing to  Carrel's  plan.  When  the  organisms  in  two  successive  counts  are  few, 
that  is,  approximately  one  per  two  fields,  and  a  culture  shows  an  absence  of 
hemolytic  cocci,  the  wound  is  considered  susceptible  of  secondary  suture 
except  when  the  wound  has  contained  hemolytic  cocci  at  any  time.  In  that 
case  careful  cultures  are  made  from  granulation  tissue  and  from  the  discharge 


708  PATHOGENIC   MICROORGANISMS 

from  all  parts  of  the  wound,  and  absence  of  hemolytie  cocci  should  be  estab- 
lished by  two  successive  negative  cultures  before  suture  is  made.  It  has  been 
observed  that  streptococci  are  prone  to  lie  dormant  in  small  numbers,  but  to 
flare  up  and  cause  virulent  infection  after  closure  of  the  wound." 

In  compound  fractures  the  same  principles  apply,  except  that, 
as  stated  by  Pool,  expedition,  thoroughness  and  early  closure  is 
particularly  important  because  it  means  the  conversion  of  open  into 
closed  fracture.  In  such  fractures  of  the  long  bones,  delayed 
primary  suture,  that  is,  suture  not  later  than  six  days  after  the 
infliction  of  the  wound,  should  be  aimed  at.  He  states  that  it  lias 
been  demonstrated  repeatedly  that  severe'  fractures  of  long  bones, 
except  the  femur,  may  be  closed  in  from  three  to  six  days  after 
debridement.  If  this  cannot  be  done,  secondary  suture  may  often 
be  made  successfully  under  proper  bacteriological  control. 

In  the  case  of  joints,  the  principle  of  treatment  consists  in  com- 
plete debridement  of  the  tract  of  the  wound  into  the  soft  parts  and 
bone,  with  the  removal  of  foreign  bodies  and  irrigation  of  the  joint, 
followed  by  absolute  closure  of  the  joint  by  suture,  with  or  without 
closure  of  the  superficial  parts. 

If  a  joint  becomes  distended  after  the  operation  and  infection 
is  suspected,  the  effusion  should  be  aspirated  and  examined  by  smear 
and  culture.  If  such  examination  indicates  infection,  the  joint 
should  be  reopened  and  treatment  for  suppurative  arthritis  begun. 

In  civil  surgery  the  principles  worked  out  with  war  wounds 
can  be  applied  with  still  greater  hope  of  success,  since  here  the 
nature  of  the  trauma  and  infection  is  apt  to  be  less  extensive. 

As  to  serological  treatment  in  civilian  surgery,  this  will  be 
applicable  chiefly  in  cases  in  which  there  has  been  a  considerable 
delay  in  the  proper  surgical  treatment  of  the  wound  after  its  inflic- 
tion. It  seems  most  probable  at  the  present  time  that  the  most 
hopeful  prospect  for  future  therapy  will  lie  in  the  combination  of 
antitoxic  sera  against  B.  Welchii,  B.  oedematiens  and  Vibrion  Sep- 
tique,  with  Tetanus  antitoxin,  prophylactically  injected  in  the  same 
way  in  which  Tetanus  antitoxin  alone  has  been  used,  hitherto. 
Whether  it  will  be  possible  to  produce  this  in  polyvalent  sera  pro- 
duced in  the  same  horse  or  whether  they  will  have  to  be  separately 
injected,  will  depend  upon  future  investigation  in  large  scale  serum 
production. 

Bacillus  of  Symptomatic  Anthrax  (Bacillus  anthracis  symp- 
tomatici,  Rauschbrand,  Ckarbon  symptomatique,  Sarcophysematos 


THE  ANAEROBIC  BACILLI  769 

bovis). — Symptomatic  anthrax  is  an  infectious  disease  occurring 
chiefly  among  sheep,  cattle,  and  goats.  It  is  spoken  of  as 
"quarter-evil"  or  "blackleg."  The  disease  has  never  been  ob- 
served in  man.  'It  was  formerly  confused  with  true  anthrax, 
because  of  a  superficial  similarity  between  the  clinical  symptoms  of 
the  two  maladies.  Bacteriologically,  the  two  microorganisms  are  in 
entirely  different  classes. 

Symptomatic  anthrax  is  of  wide  distribution  and  infection  is 
usually  through  the  agency  of  the  soil  in  which  the  bacillus  is  present, 
in  the  form  of  spores  which  may  retain  viability  .for  several  years. 


\ 


.  !  ••* 

FIG.  76. — BACILLUS  OP  SYMPTOMATIC  ANTHRAX.     After  Zettnow. 

Morphology  and  Staining. — The  bacillus  of  symptomatic  anthrax 
is  a  bacillus  with  rounded  ends,  being  about  four  to  six  micra  long, 
and  five-tenths  to  six-tenths  inicra  wide.  It  is  usually  seen  singly 
and  never  forms  long  chains.  The  bacillus  in  its  vegetative  form 
is  actively  motile  and  possesses  numerous  flagella  placed  about  its 
periphery.  In  artificial  media  it  forms  spores  which  are  oval, 
broader  than  the  rod  itself,  and  placed  near,  though  never  actually 
at,  the  end  of  the  bacillary  body.  This  gives  the  bacillus  a  racket- 
shaped  appearance. 

It  is  readily  stained  with  the  usual  anilin  dyes,  but  is  easily 
decolorized  by  Gram's  method  of  staining.  However,  von  Hibler 
claims  that  when,  very  carefully  stained  the  bacillus  can  be  shown 


770 


PATHOGENIC   MICROORGANISMS 


to  be  Grain-positive — at  least  when  taken  from  the  animal  body.32 
Cultivation. — The  bacillus  is  a  strict  anaerobe.  It  was  obtained 
in  pure  culture  first  by  Kitasato.33  Under  anaerobic  conditions  it  is 
easily  cultivated  upon  the  usual  laboratory  media,  all  of  which  are 
more,  favorable  after  the  addition  of  glucose,  glycerin,  or  nutrose. 
In  all  media  there  is  active  gas  formation,  which, 
owing  to  an  admixture  of  butyric  acid,  is  of  a  foul, 
sour  odor.  The  bacillus  is  not  very  delicate  in  its 
requirements  of  a  special  reaction  of  media,  grow- 
ing equally  well  on  those  slightly  acid  or  slightly 
alkaline. 

On  gelatin  plates,  at  20°  C.,  colonies  appear  in 
about  twenty-four  hours,  usually  round  or  oval, 
with  a  compact  center  about  which  fine  radiating 
filaments  form  an  opaque  halo.  The  gelatin  is 
liquefied. 

Surface  colonies  upon  agar  plates  are  circular 
and  made  up  of  a  slightly  granular  compact  center, 
from  which  a  thinner  peripheral  zone  emanates, 
containing  microscopically  a  tangle  of  fine  threads. 
In  agar  stabs,  at  37.5°  C.,  growth  appears  within 
eighteen  hours,  rapidly  spreading  from  the  line 
of  stab  as  a  diffuse,  fine  cloud.  Gas  formation, 
especially  near  the  bottom  of  the  tube,  rapidly  leads 
to  the  formation  of  bubbles  and  later  to  extensive 
splitting  of  the  medium.  In  gelatin  stab  cultures 
growth  is  similar  to  that  in  agar  stabs,  though  less 
FIG.  77.  —  BA-  rapid. 

CILLUS    OF    SYMP-          Pathogenicity.— Symptomatic    anthrax    bacilli 
TOMATIC  ANTHRAX. 

Culture  in  glucose     are  Path°genic  for  cattle,  sheep,  and  goats.   By  far 
agar.  the  largest  number  of  cases,  possibly  the  only  spon- 

taneous ones,  appear  among  cattle.  Guinea-pigs  are 
very  susceptible  to  experimental  inoculation.  Horses  are  very  little 
susceptible.  Dogs,  cats,  rabbits,  and  birds  are  immune.  Man  also 
appears  to  be  absolutely  immune.  Spontaneous  infection  occurs  by 
the  entrance  of  infected  soil  into  abrasions  or  wounds,  usually  of 
the  lower  extremities.-  Infection  depends  to  some  extent  upon  the 
relative  degree  of  virulence  of  the  bacillus — a  variable  factor  in  this 

32  ,'on  Hibler,  Kolle,  Wassermann,  etc,.,  p.  792,  vol.  iv. 
**Kitasaio,  Woch.  f.  Hyg,  1889. 


THE  ANAEROBIC  BACILLI  771 

species.  Twelve  to  twenty-four  hours  after  inoculation  there  appears 
at  the  point  of  entrance  a  soft,  puffy  swelling,  which  on  palpation 
is  found  to  emit  an  emphysematous  crackling.  The  emphysema 
spread  rapidly,  often  reaching  the  abdomen  and  chest  within  a  day. 
The  course  of  the  disease  is  extremely  acute,  the  fever  high,  the 
general  prostration  extreme.  Death  may  result  within  three  or 
four  days  after  inoculation. 

At  autopsy  the  swollen  area  is  found  to  be  infiltrated  with  a 
thick  exudate,  blood-tinged  and  foamy.  Subcutaneous  tissue  and 
muscles  are  edematous  and  crackle  with  gas.  The  internal  organs 
show  parenchymatous  degeneration  and  hemorrhagic  areas.  The 
bacilli,  immediately  after  death,  are  found  but  sparsely  distributed 
in  the  blood  and  internal  organs,  but  are  demonstrable  in  enormous 
numbers  in  the  edema  surrounding  the  central  focus. 

If  carcasses  are  allowed  to  lie  unburied  for  some  time,  the  bacilli 
will  attain  a  general  distribution,  and  the  entire  body  will  be  found 
bloated  with  gas,  the  organs  filled  with  bubbles.  Practically  identical 
conditions  are  found  after  experimental  inoculation. 

Toxins. — According  to  the  investigations  of  Leclainche  and 
Vallee,34  the  bacillus  of  symptomatic  anthrax  produces  a  soluble 
toxin.  It  is  not  formed  to  any  extent  in  ordinary  broth,  but  is 
formed  in  considerable  quantities  in  broth  containing  blood  or 
albuminous  animal  fluids. 

The  best  medium  for  obtaining  toxin,  according  to  the  same 
authors,  is  the  bouillon  of  Martin,35  made  up  of  equal  parts  of  veal 
infusion  and  a  pepton  solution  obtained  from  the  macerated  tissues 
of  the  stomachs  of  pigs.  The  toxin  contained  in  filtrates  of  such 
cultures  is  quite  resistant  to  heat,  but  rapidly  deteriorates  if  free 
access  of  air  is  allowed. 

Immunity. — Active  immunization  against  the  bacillus  of  symp- 
tomatic anthrax  was  first  accomplished  by  Arloing36  and  his  col- 
laborators by  the  subcutaneous  inoculation  of  cattle  with  tissue- 
extracts  of  infected  animals.  The  work  of  these  authors  resulted 
in  a  practical  method. of  immunization  which  is  carried  out  as 
follows : 

34  Leclainche  et  Vallee,  Ann.  de  1'inst.  Pasteur,  1909. 

135  Martin,  Ann.  de  1'inst.  Pasteur,  1898. 

36  Arloing,  Cornevin,  et  Thomas,  "Le  Charbon  Sympt.,"  ete.,  Paris,  1887.  Ref. 
from  Grassberger  und  Schattenfroh,  Kraus  und  Levaditi,  "Handbuch  "  etc  vol 
i,  pt.  2, 


772  PATHOGENIC   MICROORGANISMS 

Two  vaccines  are  prepared.  Vaccine  I  consists  of  the  juice  of 
infected  meat,  dried  and  heated  to  100°  C.  for  six  hours.  Vaccine  II 
is  a  similar  meat- juice  heated  to  90°  C.,  for  the  same  length  of  time. 
By  the  heating,  the  spores  contained  in  the  vaccines  are  attenuated 
to  relatively  different  degrees.  Vaccine  I  in  quantities  of  0.01  to 
0.02  c.c.  is  emulsified  in  sterile  salt  solutions  and  injected  near  the 
end  of  the  tail  of  the  animal  to  be  protected.  A  similar  quantity  of 
Vaccine  II  is  injected  in  the  same  way  fourteen  days  later. 

This  method  has  been  retained  in  principle,  but  largely  modified 
in  detail  by  various  workers.  Kitt37  introduced  the  use  of  the  dried 
and  powdered  whole  meat  instead  of  the  meat  juice,  and  made  only 
one  vaccine,  heated  to  94°  C.,  for  six  hours.  This  method  has  been 
largely  used  in  this  country.38  Passive  immunization  with  the 
serum39  of  actively  immunized  sheep  and  goats  has  been  used  in 
combination  with  the  methods  of  active  immunization. 

37  Kitt,  Ref .  from  Grassberger  und  Schattenfroh,  loc.  cit. 

38  Report  of  Bureau  of  Animal  Ind.,  Wash.,  1902. 

39  Arloing,  Leclainche  et  Vallee,  loc.  cit. 


CHAPTER    XXXVIII 
BACILLUS  ANTHRACIS  AND  ANTHRAX 

(Milzbrand,  Charbon) 

ANTHRAX  is  primarily  a  disease  of  the  hcrbivora,  attacking 
especially  cattle  and  sheep.  Infection  not  infrequently  occurs  in 
horses,  hogs,  and  goats.  In  other  domestic  animals  it  is  exceptional. 
Man  is  susceptible  to  the  disease  and  contracts  it  either  directly 
from  the  living  animals  or  from  the  hides,  wool,  or  other  parts  of 
the  cadaver  used  in  the  industries. 

The  history  of  the  disease  dates  back  to  the  most  ancient  periods 
and  anthrax  has,  at  all  times,  been  a  severe  scourge  upon  cattle- 
and  sheep-raising  communities.  Of  all  infections  attacking  the 
domestic  animals'  no  other  has  claimed  so  many  victims  as  anthrax. 
In  Russia,  where  the  disease  is  most 'common,  72,000  horses  are  said 
to  have  succumbed  in  one  year  (1864). * 

In  Austria-Hungary,  Germany,  France,  and  the  Eastern  countries, 
each  year  thousands  of  animals  and  numerous  human  beings  perish 
of  anthrax.  In  England  and  America  the  disease  is  relatively  infre- 
quent. No  quarter  of  the  globe,  however,  is  entirely  free  from  it. 

Especial  historical  interest  attaches  to  the  anthrax  bacillus  in 
that  it  was  the  first  microorganism  proved  definitely  to  bear  a  specific 
etiological  relationship  to  an  infectious  disease.  The  discovery  of 
the  anthrax  bacillus,  therefore,  laid,  as  it  were,  the  cornerstone  of 
modern  bacteriology.  The  bacillus  was  first  observed  in  the  blood 
of  infected  animals  by  Pollender  in  1849,  and,  independently,  by 
Brauell  in  1857.  Davaine,2  however,  in  1863,  was  the  first  one  to 
produce  experimental  infection  in  animals  with  blood  containing 
the  bacilli  and  to  suggest  a  direct  etiological  relationship  between 
the  two.  Final  and  absolute  proof  of  the  justice  of  Davaine 's  con- 
tentions, however,  was  not  brought  until  the  further  development 
of  bacteriological  technique,  by  Koch,3  had  made  it  possible  for 

1  Quoted  from  Sobernheim,  Kolle  und  Wassermann,  vol.  ii. 

2  Davaine,  Comptes  rend,  de  1'acad.  des.  sci.,  Ivii,  1863. 
*Koch,  Cohn's  "Beitr.  z.  Biol.  d.  Pflanzen,"  ii,  1876. 

773 


774 


PATHOGENIC   MICROORGANISMS 


this  last  observer  to  isolate  the  bacillus  upon  artificial  media  and 
to  reproduce  the  disease  experimentally  by  inoculation  with  pure 
cultures. 

Morphology  and  Staining. — The  anthrax  bacillus  is  a  straight 
rod,  5  to  10  micra  in  length,  1  to  3  micra  in  width.  It  is  non-motile. 
In  preparations  made  from  the  blood  of  an  infected  animal,  the 
bacilli  are  usually  single  or  in  pairs.  Grown  on  artificial  media  they 
form  tangles  of  long  threads.  Their  ends  are  cut  off  squarely,  in 
sharp  contrast  to  the  rounded  ends  of  many  other  bacilli,  The 


FIG.  78. — BACILLUS  ANTHRACIS.     From  pure  culture  on  agar. 

corners  are  often  sharp  and  the  ends  of  bacilli  in  contact  in  a  chain 
often  touch  each  other  only  at  these  points,  leaving  in  consequence 
an  oval  chink  between  the  ends  of  the  organisms.  The  appearance 
of  a  chain  of  anthrax  bacilli  therefore,  has  been  not  inaptly  com- 
pared to  a  rod  of  bamboo.  On  artificial  media  the  anthrax  bacillus 
forms  spores.  Oxygen  is  necessary  for  the  formation  of  these  spores 
and  they  are  consequently  not  found  in  the  blood  of  infected  sub- 
jects. The  spores  are  located  in  the  middle  of  the  bacilli  and  are 
distinctly  oval.  They  are  difficult  to  stain,  but  may  be  demonstrated 
by  any  of  the  usual  spore-staining  procedures,  such  as  Holler's  or 
Novy's  methods.  The  bacilli  themselves  are  easily  stained  by  the 
usual  anilin  dyes,  and  gentian- violet  or  fuchsin  in  aqueous  solution 


BACILLUS  ANTHRACIS  AND  ANTHRAX  775 

may  be  conveniently  employed.  They  are  not  decolorized  by  Gram's 
method. 

In  preparations  from  animal  tissues  or  blood,  stained  by  special 
procedures,  the  anthrax  bacillus  may  occasionally  be  seen  to  possess 
a  capsule.  The  capsule  is  never  seen  in  preparations  from  the 
ordinary  artificial  media.  Some  observers  have  demonstrated  them 
in  cultures  grown  in  fluid  blood  serum.  In  chains  of  anthrax  bacilli, 
the  capsule  when  present  seems  to  envelop  the  entire  chain  and  not 
the  individual  bacteria  separately. 

Isolation. — Isolation  of  the  anthrax  bacillus  from  infected  ma- 
terial is  comparatively  simple,  both  because  of  the  ease  of  its  cultiva- 
tion and  because  of  the  sharply  characteristic  features  of  its  mor- 
phological and  cultural  appearance. 

Cultivation. — The  anthrax  bacillus  is  an  aerobic,  facultatively 
anaerobic  bacillus.  While  it  may  develop  slowly  and  sparsely  under 
anaerobic  conditions,  free  oxygen  is  required  to  permit  its  luxuriant 
and  characteristic  growth. 

The  optimum  temperature  for  its  cultivation  ranges  about  37.5° 
C.  It  is. not,  however,  delicately  susceptible  to  moderate  variations 
of  temperature  and  growth  does  not  cease  until  temperatures  as  low 
as  12°  C.  or  as  high  as  45°  C.  are  reached.  By  continuous  cultivation 
at  some  of  the  temperatures  near  either  the  higher  or  the  lower  of 
these  limits,  the  bacillus  may  become  well  adapted  to  the  new 
environment  and  attain  luxuriant  growth.4 

The  anthrax  bacillus  may  be  cultivated  on  all  of  the  usual 
artificial  media,  growing  upon  the  meat-extract  as  well  as  upon  the 
meat-infusion  media. 

It  may  be  cultivated  also  upon  hay  infusion,  various  other 
vegetable  media,  sugar  solutions,  and  urine.  While  moderate  acidity 
of  the  medium  does  not  prevent  the  growth  of  this  bacillus,  the 
most  favorable  reaction  for  media  is  neutrality  or  slight  alkalinity. 

On  gelatin  plates,  colonies  develop  within  twenty-four  to  forty- 
eight  hours  as  opaque,  white  disks,  pin-head  in  size,  irregularly 
round  and  rather  flat.  As- the  colonies  increase  in  size  their  outlines 
become  less  regular  and  under  the  microscope  they  are  seen  to  be 
made  up  of  a  hair-like  tangle  of  threads  spreading  in  thin  wavy 
layers  from  a  more  compact  central  knot.  The  microscopic  appear- 
ance of  these  colonies  has  been  aptly  described  as  resembling  a 

4  Dieudonne,  Arb.  a.  d.  kais,  Gesundheitsamt,  1894. 


776  PATHOGENIC   MICROORGANISMS 

Medusa  head.  Fragments  of  a  colony  examined  on  a  slide  with  a 
higher  power  show  the  individual  threads  to  be  made  up  of  parallel 
chains  of  bacilli. 

After  a  day  or  two  of  further  growth,  the  gelatin  about  the 
colonies  becomes  liquefied. 

In  gelatin  stab  cultures,  growth  appears  at  first  as  a  thin  white  line 
along  the  course  of  the  puncture.  From  this,  growth  proceeds  in 


< 


FIG.  79. — BACILLUS  ANTHRACIS.     In  section  of  kidney  of  animal  dead  of  anthrax. 

thin  spicules  or  filaments  diverging  from  the  stab,  more  abundantly 
near  the  top  than  near  the  bottom  of  the  stab,  owing  to  more  active 
growth  in  well  oxygenated  environment.  The  resulting  picture 
is  that  of  a  small  inverted  "Christmas  tree."  Fluidification  begins 
at  the  top,  at  first  a  shallow  depression  filled  with  an  opaque  mixture 
of  bacilli  and  fluid.  Later  the  bacilli  sink  to  tlic  bottom  of  the  flat 
depression,  leaving  a  clear  supernatant  fluid  of  poptonizod  gelatin. 
In  broth,  growth  takes  place  rapidly,  but  does  not  lead  to  an  even, 
general  clouding.  There  is  usually  an  initial  pellicle  formation  at 


BACILLUS  ANTHRACIS  AND  ANTHRAX 


777 


the  top  where  the  oxygen  supply  is  greatest.  Simultaneously  with 
this  a  slimy  mass  appears  at  the  bottom  of  the  tube,  owing  to  the 
sinking  of  bacilli  to  the  bottom.  Apart  from  isolated  flakes  and 
threads  the  intervening  broth  is  clear.  Shaken  up,  the  tube  shows 
a  tough,  stringy  mass,  not  unlike  a  small  cotton  fluff,  and  general 
clouding  is  produced  only  by  vigorous  mixing. 

Upon  agar  plates,  growth  at  37.5°    C.  is  vigorous  and  colonies 
appear  within  twelve  to  twenty-four  hours.    They  are  irregular  in 


FIG.  80. — BACILLUS  ANTHBACIS.     In  smear  of  spleen  of  animal  dead  of  anthrax. 

outline,  slightly  wrinkled,  and  show  under  the  microscope  the  char- 
acteristic tangled-thread  appearance  seen  on  gelatin,  except  that 
they  are  more  compact  than  upon  the  former  medium.  The  colonies 
are  slightly  glistening  and  tough  in  consistency. 

On  agar  slants,  the  colonies  usually  become  confluent,  the  entire 
surface  soon  being  covered  by  a  grayish,  tough  pellicle  which,  if 
fished,  has  a  tendency  to  come  away  in  thin  strips  or  strands. 

On  potato,  growth  is  rapid,  white,  and  rather  dry.  Sporulation 
upon  potato  is  rapid  and  marked,  and  the  medium  is  favorable  for 
the  study  of  this  phase  of  development. 

Milk  is  slowly  acidified  and  slowly  coagulated.  This  action  is 
chiefly  upon  the  casein;  very  few,  if  any,  changes  being  produced 


778  PATHOGENIC   MICROORGANISMS 

either  in  the  sugars  or  in  the  fats  of  the  milk.  The  acids  formed 
are,  according  to  Iwanow,5  chiefly  formic,  acetic,  and  caproic  acids. 
Biological  Considerations. — The  anthrax  bacillus  is  aerobic  and 
facultatively  anaerobic.  It  is  non-motile  and  possesses  no  flagella. 
In  the  animal  body  it  occasionally  forms  capsules.  In  artificial 
cultures  in  the  presence  of  oxygen,  it  sooner  or  later  invariably 
forms  spores.  The  spores  appear  after  the  culture  has  reached  its 
maximum  of  development.  Sporulation  never  occurs  in  the  animal 
body,  probably  because  of  the  absence  of  sufficient  free  oxygen. 


FIG.  81. — ANTHRAX  COLONY  ON  GELATIN.     (After  Giinther.) 

Spores  are  formed  most  extensively6  at  temperatures  ranging  from 
20°  C.  to  30°  C.  Spore  formation  ceases  below  18°  C.  and  above 
42°  C.  For  different  strains  these  figures  may  vary  slightly,  as  has 
been  shown  from  the  results  of  various  observers.  Spores  appear 
most  rapidly  and  regularly  upon  agar  and  potato  media. 

The  spore — one  in  each  bacillus — appears  as  a  small,  highly 
refractile  spot  in  the  center  of  the  individual  bacterium.  As  this 
enlarges,  the  body  of  the  bacillus  around  it  gradually  undergoes 
granular  degeneration  and  loses  its  staining  capacity.7 

5  Iwanow,  Ann.  de  1'inst.  Pasteur,  1892. 

6  Koch,  loc.  cit. 

7  Behring,  Zeit.  f.  Hyg.,  vi  and  vii,  1889;  Dent.  med.  Woch.,  1889. 


BACILLUS  ANTHRACIS  AND  ANTHRAX  779 

If  anthrax  bacilli  are  cultivated  for  prolonged  periods  upon 
media  containing  hydrochloric  or  rosolic  acid  or  weak  solutions  of 
carbolic  acid,8  cultures  may  be  obtained  which  do  not  sporulate 
and  which  seem  permanently  to  have  lost  this  power,  without 
losing  their  virulence  to  the  same  degree.  Similar  results  may  be 
obtained  by  continuous  cultivation  at  temperatures  above  42°  C. 
By  this  procedure,  however,  virulence,  too,  is  considerably  di- 
minished. 

Resistance. — Because  of  its  property  of  spore  formation,  the 
anthrax  bacillus  is  extremely  resistant  toward  chemical  and  physical 
environment.  The  vegetative  forms  themselves  are  not  more  resist- 
ant than  most  other  non-sporulating  bacteria,  being  destroyed  by 
a  temperature  of  54°  C.  in  ten  minutes.  Anthrax  spores  may  be 
kept  in  a  dry  state  for  many  years  without  losing  their  viability.9 
While  different  strains  of  anthrax  spores  show  some  variation  in 
their  powers  of  resistance,  all  races  show  an  extremely  high  resist- 
ance to  heat.  Dry  heat  at  140°  C.  kills  them  only  after  three 
hours.10  Live  steam  at  100°  kills  them  in  five  to  ten  minutes.  Boil- 
ing in  water  destroys  them  in  about  ten  minutes.  Low  temperatures 
have  but  little  effect  upon  them.  Ravenel11  found  that,  frozen  by 
liquid  air,  they  were  still  viable  after  three  hours. 

The  variability  shown  by  different  strains  of  spores  in  their 
resistance  to  heat  is  even  more  marked  in  their  behavior  toward 
chemicals.12  Some  strains  will  retain  their  viability  after  exposure 
to  5  per  cent  carbolic  acid  for  forty  days,13  while  others  are  destroyed 
by  the  same  solution  in  two  days.  Corrosive  sublimate,  1 :2,000, 
kills  most  strains  of  anthrax  in  forty  minutes. 

Direct  sunlight  destroys  anthrax  spores  within  six  to  twelve 
hours.14 

Pathogenicity. — The  anthrax  bacillus  is  pathogenic  for  cattle, 
sheep,  guinea-pigs,  rabbits,  rats,  and  mice.  The  degrees  of  sus- 
ceptibility of  these  animals  differ  greatly,  variations  in  this  respect 
existing  even  among  different  members  of  the  same  species.  Thus, 


8  Chamberland  et  Roux,  Comptes  rend,  de  1'acad.  des  sci.,  xcvi,  1882. 

9  Surmnnt  et  Arnould,  Ann.  de  1'inst.  Pasteur,  1894. 

10  Koc.h  und  Wolff hiigel,  Mitt.  a.  d.  kais.  Gesundheitsamt,  1881. 

11  Raw'n-rl,  Medical  News,  vii,  1899. 

12  FranM,  Zeit.  f.  Hyg.,  vi,  1889. 

13  Koch,  loc.  oit. 

14  Momont,  Ann.  de  Finst.  Pasteur,  1892. 


780  PATHOGENIC   MICROORGANISMS 

the  long-haired  Algerian  sheep  show  a  high  resistance,  while  the 
European  variety  are  highly  susceptible;  and,  similarly,  the  gray 
rat  is  much  more  resistant  than  the  white  rat.  Dogs,  hogs,  cats, 
birds,  and  the  cold-blooded  animals  are  relatively  insusceptible.  For 
man  the  bacillus  is  definitely  pathogenic,  though  less  so  than  for 
some  of  the  animals  mentioned  above. 

While  separate  races  of  anthrax  bacilli  may  vary  much  in  their 
degree  of  virulence,  a  single  individual  strain  remains  fairly  constant 
in  this  respect  if  preserved,  dried  upon  threads  or  kept  in  sealed 
tubes,  in  a  cold,  dark  place.  Virulence  may  be  reduced15  by  various 
attenuating  laboratory  procedures  which  are  of  importance  in  that 
they  have  made  possible  prophylactic  immunization.  Heating  the 
bacilli  to  55°  C.  for  ten  minutes  considerably  reduces  their  virulence. 
Similar  results  are  obtained  by  prolonged  cultivation  at  tempera- 
tures of  42°  to  43°  C.,  or  by  the  addition  of  weak  disinfectants  to 
the  culture  fluids.16  Once  reduced,  the  new  grade  of  virulence 
remains  fairly  constant.  Increase  of  virulence  may  be  artificially 
produced  by  passage  through  animals. 

Experimental  infections  in  susceptible  animals  are  most  easily 
accomplished  by  subcutaneous  inoculations.  The  inoculation  is  fol- 
lowed, at  first,  by  no  morbid  symptoms,  and  some  animals  may 
appear  perfectly  well  and  comfortable  until  within  a  few  hours 
or  even  moments  before  death,  when  they  suddenly  become  visibly 
very  ill,  rapidly  go  into  collapse,  and  die.  The  length  of  the  disease 
depends  to  some  extent,  of  course,  upon  the  resistance  of  the  infected 
subject,  being  in  guinea-pigs  and  mice  from  twenty-four  to  forty- 
eight  hours.  The  quantity  of  infectious  material  introduced,  on  the 
other  hand,  has  little  bearing  upon  the  final  outcome,  a  few  bacilli, 
or  even  a  single  bacillus,  often  sufficing  to  bring  about  a  fatal 
infection.  Although  the  bacilli  are  not  demonstrable  in  the  blood 
until  just  before  death,  they  nevertheless  invade  the  blood  and 
lymph  streams  immediately  after  inoculation,  and  are  conveyed 
by  these  to  all  the  organs.  This  has  been  demonstrated  clearly  by 
experiments  where  inoculations  into  the  tail  or  ear  were  immediately 
followed  by  amputation  of  the  inoculated  parts  without  prevention 
of  the  fatal  general  infection.  The  bacilli  are  probably  not  at  first 


15  Toussaint,  Comptes  rend,  de  1'acad.  des  sci.,  xci,  1880;    Pasteur,  Chamberlan 
et  Roux,  Comptes  rend,  de  1'acad.  des  sci,  xcii,  1881. 

16  Chamberkmd  et  Rowx,  bid.,  XCVI,  1882. 


BACILLUS  ANTHRACIS  AND  ANTHRAX  781 

able  to  multiply  in  the  blood.  At  the  place  of  inoculation  and 
probably  in  the  organs  they  proliferate,  until  the  resistance  of  the 
infected  subject  is  entirely  overcome.  At  this  stage  of  the  disease, 
no  longer  held  at  bay  by  any  antagonistic  qualities  of  the  blood, 
they  enter  the  circulation  and  multiply  within  it.  Autopsy  upon 
such  animals  reveals  an  edematous  hemorrhagic  infiltration  at  the 
point  of  inoculation.  The  spleen  is  enlarged  and  congested.  The 
kidneys  are  congested,  and  there  may  be  hemorrhagic  spots  upon 
the  serous  membranes.  The  bacilli  are  found  in  large  numbers  in 
the  blood  and  in  the  capillaries  of  all  the  organs. 

The  mode  of  action  of  Bacillus  anthracis  is  as  yet  an  unsettled 
point.  It  is  probable  that  death  is  brought  about  to  a  large  extent 
by  purely  mechanical  means,  such  as  capillary  obstruction.  Neither 
a  true  secretory  toxin  nor  an  endotoxin  has  been  demonstrated  for 
the  anthrax  bacillus.  The  decidedly  toxemic  clinical  picture  of  the 
disease,  however,  in  some  animals  and  in  man,  precludes  our 
definitely  concluding  that  such  poisons  do  not  exist.  It  is  a  matter 
of  fact,  however,  that  neither  culture  filtrates  nor  dead  bacilli  have 
any  noticeable  toxic  effect  upon  test  animals,  and  exert  no  appre- 
ciable immunizing  action. 

Spontaneous  infection  of  animals  takes  place  largely  by  way  of 
the  alimentary  canal,  the  bacilli  being  taken  in  with  the  food.  The 
bacteria  are  swallowed  as  spores,  and  therefore  resist  the  acid  gastric 
juice.  In  the  intestines  they  develop  into  the  vegetative  forms, 
increase,  and  gradually  invade  the  system.  The  large  majority  of 
cattle  infections  are  of  this  type.  Direct  subcutaneous  infection 
may  also  occur  spontaneously  when  small  punctures  and  abrasions 
about  the  mouth  are  made  by  the  sharp  spicules  of  the  hay,  straw, 
or  other  varieties  of  fodder. 

When  infection  upon  a  visible  part  occurs,  there  is  formed  a 
diffuse,  tense  local  swelling,  not  unlike  a  large  carbuncle.  The 
center  of  this  may  be  marked  by  a  black,  necrotic  slough,  or  may 
contain  a  pustular  depression. 

Infection  by  inhalation  is  probably  rare  among  animals.  Trans- 
mission among  animals  is  usually  by  the  agency  of  the  excreta  or 
unburned  carcasses  of  infected  animals.  The  bacilli  escaping  from 
the  body  are  deposited  upon  the  earth  together  with  animal  and 
vegetable  matter,  which  forms  a  suitable  medium  for  sporulation. 
The  spores  may  then  remain  in  the  immediate  vicinity,  or  may  be 
scattered  by  rain  and  wind  over  considerable  areas.  The  danger 


782  PATHOGENIC   MICROORGANISMS 

from  buried  carcasses,  at  first  suspected  by  Pasteur,  is  probably 
Very  slight,  owing1  to  the  fact  that  the  bacilli  can  not  sporulate 
in  the  anaerobic  environment  to  which  the  burying-process  subjects 
them.  The  disease,  in  infected  cattle  and  sheep,  is  usually  acute, 
killing  within  one  or  two  days.  The  mortality  is  extremely  high, 
fluctuating  about  eighty  per  cent. 

In  man  the  disease  is  usually  acquired  by  cutaneous  inoculation. 
It  may  also  occur  by  inhalation  and  through  the  alimentary  tract. 

Cutaneous"  inoculation  occurs  usually  through  small  abrasions  or 
scratches  upon  the  skin  in  men  who  habitually  handle  live-stock, 
and  in  butchers,  or  tanners  of  hides.  Infection  occurs  most  fre- 
quently upon  the  hands  and  forearms.  The  primary  lesion,  often 
spoken  of  as  "malignant  pustule,"  appears  within  twelve  to  twenty- 
four  hours  after  inoculation,  and  resembles,  at  first,  an  ordinary 
small  furuncle.  Soon,  however,  its  center  will  show  a  vesicle  filled 
with  sero-sanguineous,  later  sero-purulent  fluid.  This  may  change 
into  a  black  central  necrosis  surrounded  by  an  angry  red  edematous 
areola.  Occasionally  local  gangrene  and  general  systemic  infection 
may  lead  to  death  within  five  or  six  days.  More  frequently,  how- 
ever, especially  if  prompt  excision  is  practiced,  the  patient  recovers. 
The  early  diagnosis  of  the  condition  is  best  made  bacteriologically 
by  finding  the  bacilli  in  the  local  discharge. 

The  pulmonary  infection,  known  as  "wool-sorter's  disease," 
occurs  in  persons  who  handle  raw  wool,  hides,  or  horse  hair,  by  the 
inhalation  or  by  the  swallowing  of  spores.  The  disease  is  fortunately 
rare  in  this  country.  The  spores,  once  inhaled,  develop  into  the 
vegetative  forms17  and  these  travel  along  the  lymphatics  into  the 
lungs  and  pleura.  The  disease  manifests  itself  as  a  violent,  irregular 
pneumonia,  which,  in  the  majority  of  cases,  leads  to  death.  The 
bacilli  in  these  cases  can  often  be  found  in  the  sputum  before  death. 

Infection  through  the  alimentary  canal  may  occasionally,  though 
rarely,  occur  in  man,  the  source  of  infection  being  usually  ingestion 
of  the  uncooked  meat  of  infected  animals.  This  form  of  infection 
is  rare,  because  in  many  cases  the  bacilli  have  not  sporulated  in 
the  animal  and  the  ingested  vegetative  forms  are  injured  or  de- 
stroyed by  the  acid  gastric  juice.  When  viable  spores  enter  the 
gut,  however,  infection  may  take  place,  the  initial  lesion  being 
localized  usually  in  the  small  intestine.  The  clinical  picture  that 
follows  is  one  of  violent  enteritis  with  bloody  stools  and  great 

17  Eppinger,  Wien.  med.  Woch.,  1888. 


BACILLUS  ANTHRAC1S  AND  ANTHRAX  783 

prostration.  Death  is  the  rule.  The  diagnosis  is  made  by  the  dis- 
covery of  the  bacilli  in  the  feces. 

General  hygienic  prophylaxis  against  anthrax  consists  chiefly  in 
the  destruction  of  infected  animals,  in  the  burying  of  cadavers,  and 
in  the  disinfection  of  stables,  etc.  The  practical  impossibility  of 
destroying  the  anthrax  spores  in  infected  pastures,  etc.,  makes  it 
necessary  to  resort  to  prophylactic  immunization  of  cattle  and  sheep. 

Immunity  Against  Anthrax. — Minute  quantities  of  virulent  an- 
thrax cultures  usually  suffice  to  produce  death  in  susceptible  animals. 
Dead  cultures  are  inefficient  in  calling  forth  any  immunity  in  treated 
subjects.  It  is  necessary,  therefore,  for  the  production  of  active 
immunity  to  resort  to  attenuated  cultures.  The  safest  way  to, .accom- 
plish such  attenuation  is  the  one  originated  by  Pasteur,18  consisting 
in  prolonged  cultivation  of  the  bacillus  at  42°  to  43°  C.  in  broth. 
Non-spore-forming  races  are  thus  evolved. 

The  longer  the  bacilli  are  grown  at  the  above  temperature  the 
greater  is  the  reduction  in  their  virulence.  Koch,  Gaffky,  and 
Loeffler,19  utilizing  the  variations  in  susceptibilities  of  different 
species  of  animals,  devised  a  method  by  means  of  which  the  relative 
.attenuation  of  a  given  culture  may  be  estimated  and  standardized. 
Rabbits  are  less  susceptible  than  guinea-pigs,  and  virulent  anthrax 
cultures,  grown  for  two  or  three  days  after  the  stated  conditions, 
lose  their  power  to  kill  rabbits,  but  are  less  virulent  for  guinea-pigs. 
After  ten  to  twenty  days  of  further  cultivation  at  42°  C.  the  virulence 
for  the  guinea-pig  disappears,  but  the  culture  is  potent  against  the 
still  more  susceptible  mouse.  Even  the  virulence  for  mice  may  be 
entirely  eliminated  by  further  cultivation  at  this  temperature. 

The  method  of  active  immunization  first  practiced  by  Pasteur, 
and  still  used  extensively,  is  carried  out  as  follows:  Two  anthrax 
cultures  of  varying  degrees  of  attenuation  are  used  as  vaccins.  The 
premier  vaccin  is  a  culture  which  has  lost  its  virulence  for  guinea-pigs 
and  rabbits,  and  is  potent  only  against  mice.  The  deuxieme  vaccin 
is  a  culture  which  is  still  definitely  virulent  for  mice  and  guinea-pigs, 
but  not  potent  for  rabbits.  Forty-eight-hour  broth  cultures  of  these 
strains,  grown  at  37.5°  C.,  form  the  vaccin  actually  employed.  Vaccin 
I  is  subcutaneously  injected  into  cattle  in  doses  of  0.25  c.c.,  sheep 
receiving  about  half  this  quantity.  After  twelve  days  have  elapsed 
similar  quantities  of  Vaccin  II  are  injected. 

18  Pasteur,  loc.  cit. 

19  Koch,  Goffky,  und  Loeffler,  Mitt.  a.  d.  kais.  Gesundheitsamt,  1884. 


784  PATHOGENIC   MICROORGANISMS 

Pasteur's  method  has  given  excellent  results  and  confers  an 
immunity  which  lasts  about  a  year. 

Chauveau20  has  modified  Pasteur 's  method  by  growing  the  bacilli 
in  bouillon  at  38°  to  39°  C.,  at  a  pressure  of  eight  atmospheres. 
Cultures  are  then  made  of  races  attenuated  in  this  way,  upon  chicken 
bouillon  and  allowed  to  develop  for  thirty  days.  Single  injections 
of  0.1  c.c.  each  of  such  cultures  are  said  to  protect  cattle. 

Active  immunization  of  small  laboratory  animals  is  very  difficult, 
but  can  be  accomplished  by  careful  treatment  with  extremely  at- 
tenuated cultures. 

Passive  immunization  by  means  of  the  serum  of  actively  immune 
animals  was  first  successfully  accomplished  by  Sclavo.21 

The  subject  of  passive  immunization  has  been  especially  inves- 
tigated and  practically  applied  by  Sobernheim.22  The  serum  used 
is  produced  by  actively  immunizing  sheep.  It  is  necessary  to  carry 
immunization  to  an  extremely  high  degree  in  order  to  obtain  any 
appreciable  protective  power  in  the  serum.  This  is  accomplished 
by  preliminary  treatment  with  Pasteur's  or  other  attenuated  vac- 
cines, followed  by  gradually  increasing  doses  of  fully  virulent  cul- 
tures. Treatment  continued  at  intervals  of  two  weeks,  for  two  or 
three  months,  usually  produces  an  effective  serum.  Horses  and 
cattle  may  also  be  used  for  the  process,  but  they  are  believed  by 
Sobernheim  to  give  less  active  sera  than  sheep.  Bleeding  is  done 
about  three1  weeks  after  the  last  injection.  The  sera  are  stable  and 
easily  preserved. 

Injections  of  20  to  25  c.c.  of  such  a  serum  have  been  found  to 
protect  animals  effectually  from  anthrax  and  to  confer  an  immunity 
lasting  often  as  long  as  two  months.  Animals  already  infected  are 
said  to  be  saved  by  treatment  with  25  to  100  c.c.  of  the  serum. 

Neither  specific  bactericidal  nor  bacteriolytic  properties  have, 
so  far,  been  demonstrated  in  these  immune  sera.  In  fact,  these 
properties  are  distinctly  more  pronounced  against  Bacillus  anthracis 
in  the  normal  sera  of  rats  and  dogs.  Agglutinins  have  not  been 
satisfactorily  demonstrated  in  sera,  partly  because  of  the  great 
technical  difficulties  encountered  in  the  active  chain-formation  of 
the  bacillus  in  fluid  media.  An  increase  of  opsonic  power  of  such 


20  Chauveau,  Comptos  rend,  de  1'acad.  des  sci.,  1884. 

21  Sclavo,  Cent,  f .  Bakt.,  xviii,  1895. 

22  Sobernheim,  Zeit.  f.  Hyg.,  xxv,  1897;  xxxi,  1899. 


BACILLUS  ANTHRACIS  AND  ANTHRAX 


785 


serum  over  normal  serum  has  not  been  satisfactorily  demonstrated. 
Bacteria  Closely  Resembling  Bacillus  Anthracis. — In  most  labora- 
tory collections  there  are  strains  of  true  anthrax  bacilli  so  attenuated 
that  they  are  practically  non-pathogenic.  These  do  not  differ  from 
the  virulent  strains  in  any  morphological  or  cultural  characteristics. 
Besides  such  strains  there  are  numerous  non-virulent  bacteria  cul- 


FIG.  82. — BACILLUS  SUBTILIS.     (Hay  Bacillus.) 

turally  not  identical  with  Bacillus  anthracis,  but  resembling  it  very 
closely. 

B.  ANTHRACOIDES  (Hueppe  and  Wood23). — A  Gram-positive  bacil- 
lus, morphologically  different  from  B.  anthracis  in  that  the  ends 
arc  more  rounded.  Culturally,  somewhat  more  rapid  in  growth  and 
more  rapid  in  gelatin  fluidification.  Non-pathogenic.  Otherwise 
indistinguishable  from  B.  anthracis. 

B.  RADICOSUS  (Wurzel  Bacillus). — Cultivated  from  water — city 
water  supplies.  Morphologically  somewhat  larger  than  Bacillus  an- 

23  Hueppe  und  Wood,  Berl.  klin.  Woch.,  xvi,  1889. 


786  PATHOGENIC   MICROORGANISMS 

thracis,  and  the  individual  bacilli  more  irregular  in  size.  Very  rapid 
fluidification  of  gelatin  and  growth  most  active  at  room  temperature. 
Non-pathogenic. 

B.  SUBTILIS  (Hay  Bacillus}. — Although  not  very  closely  related  to 
the  anthrax  group,  this  bacillus  is  somewhat  similar  and  conveniently 
described  in  this  connection.  It  is  of  importance  to  workers  with 
pathogenic  bacteria,  because  of  the  frequency  with  which  it  is  found 
as  a  saprophyte  or  secondary  invader  in  chronic  suppurative  lesions. 

Morphology  and  Cultivation. — Straight  rod,  2  to  8  micra  long,  0.7 
micron  wide.  Spores  formed  usually  slightly  nearer  one  pole  than  the 
other.  Grows  in  long  chains  and  only  in  such  chains  are  spores  found. 
It  does  not  decolorize  by  Gram's  method.  Is  actively  motile  in  young 
cultures  in  which  the  bacilli  are  single  or  in  pairs.  In  older  cultures 
chains  are  formed  and  the  bacilli  become  motionless.  Gelatin  is 
liquefied.  On  gelatin  and  agar  the  bacilli  grow  as  a  dry  corrugated 
pellicle.  Microscopically,  the  colonies  are  made  up  of  interlacing 
threads,  being  ^irregularly  round  with  fringed  edges.  There  is  a 
tendency  to  confluence.  !  The  bacillus  is  found  in  brackish  water, 
infusions  of  vegetable  matter,  etc.,  and  is  practically  non-pathogenic, 
occurring  only  occasionally  as  a  saprophyte  in  old  sinuses  and 
infected  wounds.  * 


CHAPTER   XXXIX 
BACILLUS   MALLEI  AND   GLANDERS 

(Glanders  Bacillus) 

GLANDERS  is  an  infectious  disease  prevalent  chiefly  among  horses, 
but  transmitted  occasionally  to  other  domestic  animals  and  to  man. 
The  microorganism  causing  the  disease,  though  seen  and  described 
by  several  earlier  authors,  was  first  obtained  in  pure  culture  and 
accurately  studied  by  Loeffler  and  Schiitz1  in  1882. 

Morphology  and  Staining. — The  glanders  bacillus  or  B.  mallei 
is  a  rather  small  rod  with  rounded  ends.2  Its  length  varies  from 
3  to  4  micra,  its  breadth  from  0.5  to  0.75  micron.  Variation  in  size 
between  separate  individuals  in  the  same  culture  is  characteristic. 
The  rods  are  usually  straight,  but  may  show  a  slight  curvature. 
The  bacillus  is  non-motile.  There  are  no  flagella  and  no  spores  are 
formed.  The  grouping  of  the  bacilli  in  smears  shows  nothing  very 
characteristic.  Usually  they  appear  as  single  bacilli  lying  irregularly 
parallel,  often  in  chains  of  two  or  more.  In  old  cultures,  involution 
forms  appear  which  are  short,  vacuolated,  and  almost  coccoid. 

While  the  glanders  bacillus  stains  rather  easily  with  the  usual 
anilin  dyes,  it  is  so  easily  decolorized  that  especial  care  in  preparing 
specimens  must  be  observed.  Stained  in  the  usual  manner  with 
methyleiie-blue,  it  shows  marked  irregularity  in  its  staining  quali- 
ties; granular,  deeply  staining  areas  alternating  with  very  faintly 
stained  or  entirely  unstained  portions.  This  diagnostically  helpful 
characteristic  has  been  variously  interpreted  as  a  mark  of  degenera- 
tion or  a  preparatory  stage  for  sporulation.  It  is  probably  neither 
of  the  two,  but  an  inherent  irregularity  in  the  normal  protoplasmic 
composition  of  the  bacillus,  not  unlike  that  of  B.  diphtherias.  The 
bacillus  is  decolorized  by  Gram's  method  of  staining. 

Cultivation. — The  glanders  bacillus  is  easily  grown  on  all  of  the 
usual  meat-infusion  media.  It  is  practically  indifferent  to  moderate 


1  Loeffler  und  Schutz,  Deut.  med.  Woch.,  1882. 

2  Loeffler,  Arb.  a.  d.  kais.  Gesundheitsamt,  1886. 

787 


788  PATHOGENIC  MICROORGANISMS 

variations  in  reaction,  growing  equally  well  upon  neutral,  slightly 
acid,  or  slightly  alkaline  culture  media.  Glycerin  or  small  quantities 
of  glucose  added  to  media  seem  to  render  them  more  favorable  for 
the  cultivation  of  this  bacillus. 

Upon  agar  the  colonies  show  little  that  is  characteristic.  They 
appear  after  twenty-four  hours  at  37.5°  C.  as  yellowish-white  spots, 
at  first  transparent,  later  more  opaque.  They  are  round,  with  an 
even  border,  and  microscopically  appear  finely  granular.  The  older 
the  cultures  are,  the  more  yellow  do  they  appear. 

On  gelatin  at  room  temperature,  growth  is  slow,  grayish-white,  and 


FIG.  83.  —  GLANDERS  BACILLUS.     From  potato  culture.     (After  Zettnow.) 

no  liquefaction  of  the  gelatin  occurs.  Growth  upon  this  medium 
is  never  abundant. 

In  broth,  there  is,  at  first,  diffuse  clouding,  later  a  heavy,  tough, 
slimy  sediment  is  formed.  At  the  same  time  the  surface  is  covered 
with  a  similarly  slimy  pellicle.  The  broth  gradually  assumes  a  dark 
brown  color. 

In  milk,  coagulation  takes  place  slowly.  In  litmus  milk,  acidifica- 
tion appears. 

The  growth  upon  potato  presents  certain  features  which  are  diag- 
nostically  valuable.  On  potatoes  which  are  not  too  acid  growth 
is  abundant  and  within  forty-eight  hours  covers  the  surface  as  a 
yellowish,  transparent,  slimy  layer.  This  gradually  grows  darker 


BACILLUS  MALLEI  AND  GLANDERS  789 

until  it  has  assumed  a  deep  reddish-brown  hue.  In  using  this  feature 
of  the  growth  diagnostlcally,  it  must  not  be  forgotten  that  a  very 
similar  appearance  upon  potato  occurs  in  the  case  of  B.  pyocyaneus. 

Biological  Considerations. — Bacillus  mallei  is  aerobic.3  Growth 
under  anaerobic  conditions  may  take  place,  but  it  is  slow  and 
impoverished.  The  most  favorable  temperature  for  its  cultivation 
is  37.5°  C.  It  fails  to  develop  at  temperatures  below  22°  C.  or  above 
43°  C.  On  artificial  media,  if  kept  cool  and  in  the  dark,  and  in 
sealed  tubes,  the  glanders  bacillus  will  retain  its  viability  for  months 
and  years.  On  gelatin  and  in  bouillon,  it  lives  for  a  longer  time 
than  on  the  other  media.  Exposed  to  strong  sunlight  it  is  killed 
within  twenty-four  hours.  Heating  to  60°  C.  kills  it  in  two  hours, 
to  75°  C.  within  one  hour.  Thorough  drying  kills  the  glanders 
bacillus  in  a  short  time.  In  water,  under  the  protected  conditions 
that  are  apt  to  prevail  in  watering-troughs,  the  bacillus  may  remain 
alive  for  over  seventy  days.  The  resistance  to  chemical  disinfectants 
is  not  very  high.4  Carbolic  acid,  one  per  cent,  kills  it  in  thirty 
minutes,  bichlorid  of  mercury,  0.1  per  cent,  in  fifteen  minutes. 

Pathogenicity. — Spontaneous  infection  with  the  glanders  bacillus 
occurs  most  frequently  in  horses.  It  occurs  also  in  asses,  in  cats, 
and,  more  rarely,  in  dogs.  In  man  the  disease  is  not  infrequent 
and  is  usually  contracted  by  those  in  habitual  contact  with  horses. 
Experimental  inoculation  is  successful  in  guinea-pigs  and  rabbits. 
Cattle,  hogs,  rats,  and  birds  are  immune  to  experimental  and  spon- 
taneous infections  alike. 

Spontaneous  infection  takes  place  by  entrance  through  the 
broken  skin,  through  the  mucosa  of  the  mouth  or  nasal  passages. 
Infection  in  horses  not  infrequently  takes  place  through  the  digestive 
tract.5  In  all  cases,  so  far  as  we  know,  previous  injury  to  either 
the  skin  or  to  the  mucosa  is  necessary  for  penetration  of  the  bacilli 
and  the  development  of  the  disease. 

Glanders  in  horses  may  occur  in  an  acute  or  chronic  form, 
depending  upon  the  relative  virulence  of  the  infecting  culture  and 
the  susceptibility  of  the  subject.  The  more  acute  form  of  the  disease 
is  usually  limited  to  the  nasal  mucosa  and  upper  respiratory  tract. 
The  more  chronic  type  of  the  disease  is  often  accompanied  by  mul- 


3  Loeffler,  loc.  cit. 

4  Finger,  Ziegler's  Beitr.,  vi,  1889. 

5  Nocard,  Bull,  de  la  soc.  centr.  de  med.  vet.,  1894. 


790  PATHOGENIC   MICROORGANISMS 

tiple  swellings  of  the  skin  and  general  lymphatic  enlargement.    This 
form  is  often  spoken  of  as  *  'farcy." 

Acute  glanders  in  the  horse  begins  violently  with  fever  and 
prostration.  After  two  or  three  days  there  is  a  nasal  discharge, 
at  first  serous,  later  seropurulent.  At  the  same  time  there  is  ulcera- 
tion  of  the  nasal  mucosa  and  acute  swelling  of  the  neighboring 
lymph  nodes.  These  may  break  down  and  form  deep  pus-discharging 


V- 

V    i     v 


FIG.  84. — GLANDERS  BACILLI  IN  TISSUE.     (From  a  drawing  furnished  by 
Dr.  James  Ewing.) 

sinuses  and  ulcers.    Finally,  there  is  involvement  of  the  lungs  and 
death  within  four  to  six  weeks. 

When  the  disease  takes  the  chronic  form  the  onset  is  more 
gradual.  Concomitant  with  the  nasal  inflammation  there  is  a  forma- 
tion of  subcutaneous  swellings  all  over  the  body,  some  of  which 
show  a  tendency  to  break  down  and  ulcerate.  Together  with  this 
the  lymphatics  all  over  the  body  become  enlarged.  The  disease  may 
last  for  several  years,  and  occasionally  may  end  in  complete  cure. 


BACILLUS   MALLEI   AND   GLANDERS  791 

In  horses  the  chronic  form  of  the  disease  is  by  far  the  more  frequent. 
In  man  the  disease  is  similar  to  that  of  the  horse  except  that  the 
point  of  origin  is  more  frequently  in  some  part  of  the  skin  rather 
than  in  the  nasal  mucosa,  and  the  clinical  symptoms  differ  accord- 
ingly. The  onset  is  usually  violent,  with  fever  and  systemic  symp- 
toms. At  the  point  of  infection  a  nodule  appears,  surrounded  by 
lymphangitis  and  swelling.  A  general  papular  eruption  may  occur. 
The  papules  may  become  pustular,  and  the  clinical  features  may 
thus  simulate  variola.  This  type  of  the  disease  usually  ends  fatally 
in  eight  to  ten  days.  The  chronic  form  of  the  disease  in  man  is 
much  like  that  in  the  horse,  but  is  more  frequently  fatal. 

The  histological  appearance  of  the  glanders  nodules  is  usually 
one  of  diffuse  leucocytic  infiltration  and  the  formation  of  young 
connective  tissue  which  preponderates  more  and  more  as  the  disease 
becomes  chronic.  Virchow  has  classed  these  lesions  with  the  granu- 
lomata.  From  the  center  of  such  nodules  B.  mallei  may  often  be 
obtained  in  pure  culture.  The  nodules  may  be  generally  distributed 
throughout  the  internal  organs.  The  bacilli  themselves  are  found, 
apart  from  the  nodules,  in  the  nasal  secretions,  and  occasionally  in 
the  circulating  blood.6 

The  bacteriological  diagnosis  of  glanders  may  be  made  by  isolating 
and  identifying  the  bacilli  from  any  of  the  above-mentioned  sources. 
When  superficial  nodules  can  be  opened  for  the  purpose  of  diagnosis 
this  may  prove  an  easy  task.  The  most  diagnostically  helpful 
medium  in  such  cases  is  potato.  In  a  majority  of  cases,  however, 
isolation  is  extremely  difficult  and  resort  must  be  had  to  animal 
inoculation.  The  most  suitable  animal  for  this  purpose  is  the  male 
guinea-pig.  Intraperitoneal  inoculation  of  such  animals  with  ma- 
terial containing  glanders  bacilli  leads  within  two  or  three  days 
to  tumefaction  and  purulent  inflammation  of  the  testicles.  Such 
an  experiment,  spoken  of  as  the  * '  Strauss  test, ' ' 7  should  always 
be  reinforced  by  cultural  examination  of  the  testicular  pus,  the 
spleen,  and  the  peritoneal  exudate  of  the  animals  employed. 

Toxin  of  Bacillus  mallei. — The  toxin  of  B.  mallei,  or  mallein, 
belongs  to  the  class  of  endotoxins.  The  toxic  products  have  been 
invariably  obtained  by  extraction  of  dead  bacilli.8  Mallein  differs 


6  Wassilieff,  Deut.  med.  Woch.,  1883. 
''Strauss,  Arch,  de  mcd.  exp.,  1889. 


*Kresling,  Arch.  d.  sci.  biol.,  1892;   Preuser,  Berl.  thierarzt.  Woch.,  1894. 


792  PATHOGENIC   MICROORGANISMS 

from  many  other  bacterial  poisons  in  being  extremely  resistant.  It 
withstands  temperatures  of  120°  C.  and  prolonged  storage  without 
noticeable  loss  of  strength.9 

In  its  physiological  action  upon  healthy  animals,  mallein  is  not 
a  powerful  poison.  It  can  be  given  in  considerable  doses  without 
causing  death.  Mallein  may  be  obtained  by  a  variety  of  methods. 
Helman  and  Kalning,  the  discoverers  of  this  toxin,  used  filtered  aque- 
ous and  glycerin  extracts  of  potato  cultures.  Houx10  cultivates 
virulent  glanders  bacilli  in  flasks  containing  250  c.c.  each  of  5  per 
cent  glycerin  bouillon.  Growth  is  allowed  to  continue  at  35°  C. 
for  one  month.  At  the  end  of  this  time,  the  cultures  are  sterilized 
at  100°  for  thirty  minutes,  and  evaporated  on  a  water  bath  to  one- 
tenth  their  original  volume.  They  are  then  filtered  through  paper. 
This  concentrated  poison  is  diluted  ten  times  with  0.5  per  cent 
carbolic  acid  before  use.  Concentration  is  done  merely  for  purposes 
of  conservation.  The  diagnostic  dose  of  such  mallein  for  a  horse 
is  0.25  c.c.  of  the  undiluted  fluid. 

At  the  Washington  Bureau  of  Animal  Industry,  mallein  is  pre- 
pared by  growing  the  bacilli  for  five  months  at  37.5°  C.  in  glycerin- 
bouillon.  This  is  then  boiled  for  one  hour  and  allowed  to  stand 
in  a  cool  place  for  one  week.  The  supernatant  fluid  is  then  decanted 
and  filtered  through  clay  filters  by  means  of  a  vacuum  pump.  The 
filtrate  is  evaporated  to  one-third  its  original  volume  on  a  water 
bath,  and  the  evaporated  volume  resupplied  by  a  1  per  cent  carbolic 
acid  solution  containing  about  10  per  cent  of  glycerin. 

Diagnostic  Use  of  Mallein. — The  injection  of  a  proper  dose  of 
mallein  into  a  horse  suffering  from  glanders  is  followed  within  six 
to  eight  hours  by  a  sharp  rise  of  temperature,  often  reaching  104° 
to  106°  F.  (40°  C.  +).  The  high  temperature  continues  for  several 
hours  and  then  begins  gradually  to  fall.  The  normal  is  not  usually 
regained  for  several  days.  Locally,  at  the  point  of  injection,  there 
appears  Within  a  few  hours  a  firm,  hot,  diffuse  swelling,  which 
gradually  extends  until  it  may  cover  areas  of  20  to  30  centimeters 
in  diameter.  The  swelling  is  intensely  tender  during  the  first  twenty- 
four  hours,  and  lasts  for  three  to  nine  days.  Together  with  this 
there  are  marked  symptoms  of  general  intoxication.  In  normal 
animals  the  rise  of  temperature  following  an  injection  is  trifling, 

9  Wladimiroff,  in  Kraus  und  Levaditi,  "Handbuch,"  etc.,  1908. 
lnRoux  et  Nocard,  Bull.  d.  1.  soc.  centr.  v6t.,  1892. 


BACILLUS   MALLEI  AND  GLANDERS  793 

and  the  local  reaction  is  much  smaller  and  more  transient.  Injec- 
tions are  best  made  into  the  breast  or  the  side  of  the  neck. 

The  directions  given  by  the  United  States  Government  for  using 
mallein  for  the  diagnosis  of  glanders  in  horses  are  as  follows : 

'  *  Make  the  test,  if  possible,  with  a  healthy  horse,  as  well  as  with 
one  or  more  affected  or  supposed  to  be  affected  with  glanders.  Take 
the  temperature  of  all  these  animals  at  least  three  times  a  day  for 
one  or  more  days  before  making  the  injections. 

"The  injection  is  most  conveniently  made  at  6  or  7  o'clock  in 
the  morning,  and  the  maximum  temperature  will  then  usually  be 
reached  by  or  before  10  P.M.  of  the  same  day. 

"Use  for  each  horse  one  cubic  centimeter  of  the  mallein  solution 
as  sent  out,  and  make  the  injection  beneath  the  skin  of  the  middle 
of  one  side  of  the  neck,  where  the  local  swelling  can  be  readily 
detected. 

"Carefully  sterilize  the  syringe  after  injecting  each  horse  by 
naming  the  needle  over  an  alcohol  lamp  or,  better,  use  separate 
syringes  for  healthy  and  suspected  animals.  If  the  same  syringe 
is  used,  inject  the  healthy  animals  first,  and  flame  the  needle  of 
the  syringe  after  each  injection. 

"Take  the  temperature  every  two  hours  for  at  least  eighteen 
hours  after  the  injection.  Sterilize  the  thermometer  in  a  5  per  cent 
solution  of  carbolic  acid,  or  a  0.2  per  cent  solution  of  corrosive 
sublimate,  after  taking  the  temperature  of  each  animal. 

"The  temperature,  as  a  rule,  will  begin  to  rise  from  four  to  eight 
hours  after  the  injection,  and  reach  its  maximum  from  ten  to  sixteen 
hours  after  injection.  On  the  day  succeeding  the  injection  tako 
the  temperature  at  least  three  times. 

"In  addition  to  the  febrile  reaction,  note  the  size,  appearance, 
and  duration  of  any  local  swelling  at  the  point  of  injection.  Note 
the  general  condition  and  symptoms  of  the  animal,  both  before, 
during,  and  after  the  test. 

"Keep  the  solution  in  the  sealed  bottle  and  in  a  cool  place,  and 
do  not  use  it  when  it  is  clouded  or  if  it  is  more  than  six  weeks 
old ;  when  it  leaves  the  laboratory  of  the  Bureau  it  is  sterile. ' ' 

If  the  result  of  first  injection  is  doubtful,  the  horse  should  be 
isolated  and  retested  in  from  one  to  three  months,  when  the  slight 
immunity  conferred  by  the  first  injection  will  have  disappeared. 
The  second  injection  into  healthy  horses  usually  shows  no  reaction 
whatever. 


794  PATHOGENIC   MICROORGANISMS 

Mallein  may  cause  reactions  in  the  presence  of  other  diseases 
than  glanders,  such  as  bronchitis,  periostitis,  and  other  inflammatory 
lesions  and  is  not  so  specifically  valuable  as  tuberculin  for  diagnosis. 

Complement  Fixation  in  Glanders. — Diagnostic  complement  fixa- 
tion for  the  diagnosis  of  glanders  has  been  developed  by  McNeil 
and  Olmstead  at  the  New  York  Department  of  Health.  The  antigen 
is  made  by  growing  the  glanders  bacilli  on  a  1.6  per  cent  glycerin 
potato  agar.  From  this  stock  cultures  transplanted  are  made  upon 
a  neutral  meat-free-veal-peptone  agar.  Twenty-four-hour  growths 
are  washed  off  with  distilled  water  sterilized  at  80°  C.  for  four  hours 
and  filtered  through  a  Berkefeld.  After  filtration  the  antigen  must 
again  be  sterilized  at  80°  for  one  hour. 

Immunity. — Recovery  from  a  glanders  infection  does  not  confer 
immunity  against  a  second  inoculation.11  Artificial  active  immuniza- 
tion has  been  variously  attempted  by  treatment  with  attenuated 
cultures,  with  dead  bacilli,  and  with  mallein,  but  without  convincing 
results. 

The  serum  of  subjects  suffering  from  glanders  contains  specific 
agglutinins.12  These  are  of  great  importance  diagnostically  if  the 
tests  are  made  with  dilutions  of,  at  least,  1  in  500,  since  normal 
horse  serum  may  agglutinate  B.  mallei  in  dilutions  lower  than  this. 

11  Finger,  Ziegler's  Beitrage,  vi,  1899. 

12  Galtier,  Jour,  de  med.  vet.,  1901. 


CHAPTER    XL 

THE  BACILLUS  MELITENSIS  (MICROCOCCUS  MELITENSIS),  BACILLUS 
BRONCHISEPTICUS,  BACILLUS  OF  CATTLE  ABORTION,  BACILLUS 
OF  GUINEA-PIG  PNEUMONIA,  AND  THE  BACILLUS  PYOCYANEUS. 

MALTA  FEVER  AND  THE  BACILLUS  MELITENSIS 

MALTA  fever  probably  has  its  endemic  focus  in  the  Mediterranean 
islands  and  along  the  Mediterranean  coast.  From  here  it  appears 
to  have  spread  into  continental  Europe,  France,  Italy  and  Spain 
and  into  the  Balkans.  Naturally  enough  it  has  been  found  to  occur 
in  Northern  Africa  and  cases  have  been  reported  along  the  East 
African  coast.  Castellani  and  Chalmers1  state  that  it  has  also  been 
found  in  parts  of  Russia,  in  South  Africa,  in  Uganda,  China  and 
the  Philippines  and  in  North  and  South  America  and  in  the  West 
Indies.  Castellani  also  has  reported  cases  from  Ceylon. 

It  appears,  thus,  that  the  disease  is  very  widely  distributed,  but 
centralizes  chiefly  about  the  Mediterranean,  being  most  common  in 
the  warmer  temperate  and  subtropical  climates.  Studies  on  its  dis- 
tribution have  been  made  particularly  by  Bassett-Smith,2  who  has 
mapped  its  distribution  throughout  the  world.  A  reproduction  of 
his  map  may  be  found  in  Castellani  and  Chalmer's  book. 

Morphology. — The  micrococcus  melitensis  is  an  extremely  small 
bacterium  which  has  been  described  both  as  a  bacillus  and  a  coccus. 
It  has  been  the  custom  of  bacteriologists  who  have  studied  it  more 
recently  to  regard  it  as  a  bacillus  and  speak  of  it  as  the  Bacillus 
Melitensis.  Eyre3  describes  it  as  an  extremely  small  coccus  which 
has  bacillary  forms  on  various  media  which  he  regards  as  involution 
forms.  It  is  Gram-negative  and  non-motile  and  does  not  form  spores. 
In  cultures  it  appears  both  singly  and  in  short  chains  of  two  or 
more.  Chains  of  any  considerable  length  are  uncommon. 

1  Castellani  and  Chalmers,  Textbook  of  Tropical  Medicine,  William  Wood  &  Co., 
N.  Y.,  1919. 

2  Bassett-Smith,  British  Med.  Jour.,  2,  1904. 

3  Eyre,  Kolle  and  Wassermann  Handb.,  Second  Edition,  Vol.  4,  421. 

795 


796  PATHOGENIC   MICROORGANISMS 

Cultivation. — The  organism  can  usually  be  cultivated  from  the 
spleens  of  those  who  have  died  of  the  disease,  or  by  spleen  puncture, 
a  method  by  which  Bruce4  obtained  it  in  his  early  studies.  It  can 
also  be  obtained  from  the  blood  stream  in  active  cases,  from  the 
urine  and  from  the  milk  of  infected  goats.  Eyre  states  that  the 
optimum  is  at  37°  C.  and  that  it  will  grow  but  slightly  and  slowly 
on  media  at  room  temperature  at  20°  or  thereabout.  It  will  grow 
both  aerobically  and  under  conditions  of  limited  anaerobiosis. 
Growth  is  relatively  slow  and  does  not  become  luxuriant  for  three 
days  or  more.  It  does  not  seem  to  be  very  delicate  in  its  nutritive 
requirements  and  has  been  cultivated  on  most  of  the  ordinary  media. 
It  will  grow  on  gelatin  without  liquefying  the  gelatin.  Its  growth 
on  potato  is  hardly  visible. 

Animal  Pathogenicity. — According  to  Eyre,  the  B.  melitensis  is 
pathogenic  for  almost  all  laboratory  animals,  although  it  may  take 
a  very  long  time  to  kill.  Eyre  states  that  guinea  pigs  will  live 
for  as  long  as  100  or  more  days  after  an  injection  of  B.  melitensis, 
but  that  the  virulence  of  the  organism  can  be  greatly  enhanced  by 
animal  passage.  It  spontaneously  infects  goats  which,  as  we  shall 
see,  is  an  important  point  in  its  epidemiology,  and  apparently  it  may 
similarly  infect  horses,  cattle  and  sheep. 

The  Disease. — In  a  number  of  ways,  Malta  fever  is  similar  to 
typhoid  fever.  It  probably  gets  into  man  in  most  cases  by  way  of 
mouth,  passing  from  the  mucous  membrane  of  the  intestinal  canal 
into  the'  blood  without  causing  any  considerable  lesions.  A  bac- 
teriemia  follows  during  which  there  is  a  typhoid  fever-like  tempera- 
ture and  an  enlarged  spleen.  The  incubation  time  seems  to  be  about 
two  weeks,  and  the  onset  of  the  disease  in  its  generalized  symptoms 
again  has  the  indefinite  characters,  malaise,  headache,  etc.,  that  are 
associated  with  typhoid.  There  is  no  leucocytosis  and  a  relative 
lymphocytosis.  Usually  cases  are  protracted,  the  disease  passing 
through  a  prolonged  febrile  period,  lasting  two,  three,  or  four  weeks. 
There  may  be  acute  cases  in  which  the  onset  is  sudden  and  the 
course  of  the  disease  violent. 

Secondary  symptoms  may  consist  in  neuritis,  parenchymatous 
nephritis  and  pulmonary  congestion,  arthritis  and  orchitis. 

4  Bruce,  Practitioner,  1887. 


THE  BACILLUS   MELITENSIS  797 

Diagnosis  can  be  made  by  agglutination  and  isolation  of  the 
organisms.  According  to  Wright  and  Semple,5  Bassett-Smith,  Eyre6 
and  others,  the  agglutinins  in  the  blood  are  very  high  and  may  be 
detected  in  dilutions  of  1 :1,000  or  over,  even  as  early  as  the  end 
of  the  first  week. 

Blood  cultures  may  be  positive  very  early  in  the  disease.  Later, 
the  organisms  appear  in  the  urine. 

Epidemiology. — It  seems  to  be  unquestionable  at  the  present  time 
that  the  disease  is  transmitted  chiefly  by  the  ingestion  of  the  milk 
of  infected  goats,  and  according  to  Eyre  the  goat  is  the  natural 
host  of  the  infection,  keeping  the  disease  going  in  endemic  regions. 
Apparently  a  very  large  percentage  of  the  goats  in  infected  regions 
are  infected.  According  to  Castellani  and  Chalmers  in  Malta  50 
per  cent  of  the  goats  are  infected,  and  in  parts  of  Northern  Africa 
the  percentage  of  infection  in  goats  as  indicated  by  various  re- 
searches ranges  from  less  than  4  to  34  per  cent. 

Since  goats'  milk  is. a  common  human  food  in  these  regions,  the 
spread  of  the  disease  by  this  means  is  natural. 

There  are  probably  carriers  among  goats  and  human  beings  that 
recover.  Among  other  forms  of  transmission,  direct  infection  from 
individual  to  individual  probably  takes  place,  and  indirect  infection 
through  food  and  flies  is  not  out  of  question. 

It  has  been  suggested  that  mosquitoes,  too,  may  transmit  the 
disease  and  Eyre  states  that  it  has  been  possible  to  demonstrate 
the  organisms  in  the  stomachs  of  a  number  of  different  mosquito 
species. 

However,  it  seems  quite  clear  that  the  most  important  method 
of  transmission  is  by  means  of  infected  milk. 

Immunity. — It  is  the  general  belief  that  one  attack  of  Malta 
fever  protects  against  a  second  attack.  However,  there  are  a  great 
many  cases  in  which  the  fever  recurs  several  times  in  the  same 
individual  in  the  form  of  relapses  and  recrudescences.  The  inter- 
missions between  such  attacks  often  last  for  months.  Prophylactic 
vaccination  has  not  yet  been  carried  out  on  a  sufficiently  large  scale 
to  permit  the  formulation  of  conclusions. 


5  Wright  and  Semple,  Lancet,  656,  1897. 

6  Eyre,  etc.,  Report  of  the  Mediterranean  Fever  Commission,  1907. 


798  PATHOGENIC   MICROORGANISMS 

B.  Bronchiseptisus. — Prior  to  1910  a  great  deal  of  inconclusive 
work  was  done  on  canine  distemper,  but  in  this  year  Ferry7  published 
a  preliminary  paper  in  which  he  reported  the  isolation  of  a  bacillus 
which  was  often  present  in  pure  culture  in  the  smaller  bronchii  and 
the  tracheae  of  dogs  killed  early  in  the  disease.  At  almost  the 
same  time,  and  independently  of  Ferry,  McGowan8  reported  similar 
observations.  Soon  after  this  the  subject  was  very  thoroughly 
studied  by  Torrey  and  Rahe.9  The  B.  bronchisepticus  was  described 
as  a  short,  Gram-negative  organism,  occasionally  coccoid  in  appear- 
ance, slowly  motile,  which  grows  very  slowly  at  first  isolation,  the 
colonies  being  hardly  visible  on  agar  in  twenty-four  hours  but 
definitely  visible  in  forty-eight  hours.  It  grows  well  on  glycerin 
agar,  will  grow  at  20°  with  an  optimum  at  about  37.5°.  It  is  not 
an  obligate  aerobe,  but  grows  poorly  without  oxygen.  It  does  not 
liquefy  gelatin.  It  renders  broth  uniformly  turbid  with  a  slight 
deposit  and  no  pellicle.  No  acid  or  gas  is  formed  on  carbohydrate 
media,  and  the  media  are  rendered  slightly  alkalin  in  the  course  of 
four  or  five  days.  Torrey  could  determine  no  indol  formation.  The 
typical  media,  according  to  McGowan,  Ferry  and  Torrey  are  litmus 
milk  and  potato.  Torrey  states  that  the  use  of  these  two  media 
alone  is  sufficient  for  identification  in  the  hands  of  a  practiced 
observer. 

On  litmus  milk  it  grows  like  the  B.  fecalis  alkaligenes.  There 
is  a  progressive  alkalinization,  and  after  about  twenty-four  hours 
at  37°  a  ring  of  deep  blue  appears,  extending  about  3/8  inch  from 
the  surface.  In  from  five  to  ten  days  the  whole  medium  has  assumed 
a  blue-black  color.  On  potato  in  twenty-four  hours  a  marked  yel- 
lowish-brown growth  appears  with  sometimes  a  greenish  darkening 
of  the  potato.  In  this  respect  again  it  is  very  similar  to  B.  fecalis 
alkaligenes.  Torrey  also  reports  that  the  organism  produces  a 
hemolysin  for  rabbit  and  guinea  pig  erythrocytes.  He  states 
that  typical  distemper  can  be  induced  in  susceptible  dogs  by  injec- 
tion with  pure  cultures,  and  dogs  which  have  recovered  from  attacks 
induced  by  the  bacillus  are  protected  by  exposure  to  the  disease  in 
other  dogs. 


7  Ferry,  Amer.  Veterinary  Review,  499,  1910  (quoted  from  Torrey)  Jour.  Infec 
Dis.,  8,  1911,  399. 

8  McGowan,  Jour.  Path,  and  Bacter.,  15,  1911,  372  and  1916,  257, 

9  Torrey  and  Rahe,  Jour.  Med.  Res.,  27,  1912,  291. 


THE   BACILLUS   MELITENSIS  799 

Bacillus  of  Cattle  Abortion  (B.  Abortus  of  Bang).— We  have 
already  mentioned,  in  speaking  of  the  organism  which  causes  abor- 
tion in  mares,  that  there  was  another  bacillus  described  by  Bang10 
in  1897  which  was  found  by  him  to  be  the  etio logical  factor  in 
abortion  of  cattle,  but  which  was  distinctly  different  from  that 
subsequently  found  by  Smith  and  others  in  the  analogous  disease 
of  horses.  This  organism  has  been  observed  by  a  great  many  writers 
since  Bang,  and  it  was  Smith  who  first  pointed  out  its  similarity 
to  the  B.  bronchisepticus  of  canine  distemper  and  to  a  bacillus 
which  causes  epidemic  pneumonias  in  guinea  pigs.  Alice  Evans11 
subsequently  pointed  out  the  similarity  of  B.  Abortus  and  of  B. 
bronchisepticus,  to  the  B.  melitensis  which  causes  Malta  fever. 
Evans'  description  of  Bacterium  Abortus  as  studied  from  strains 
obtained  by  the  Dairy  Division  of  the  Bureau  of  Animal  Industry, 
United  States  Department  of  Agriculture,  is  as  follows : 

It  is  a  short,  slender  pleomorphic  rod  with  rounded  ends,  some- 
times so  short  as  to  appear  coccoid.  Obtained  from  the  condensa- 
tion water  of  a  twenty-four  hour  culture  on  agar,  it  is  non-motile 
and  does  not  form  spores.  It  is  Gram-negative. 

It  is  difficult  to  grow  on  artificial  media  on  first  isolation,  and 
in  these  early  cultures  its  growth  is  favored  by  partial  anaerobiosis, 
which  Evans  obtained  by  incubation  in  a  closed  jar  in  the  presence 
of  cultures  of  B.  subtilis.  Glycerin  or  serum  agar  are  favorable 
media  for  isolation,  but  after  prolonged  cultivation  it  grows  well 
on  ordinary  media.  Colonies  on  agar  plates  develop  after  about 
two  days  in  very  small  dewdrop  form.  It  never  heavily  clouds 
broth.  Milk  is  rendered  slightly  alkaline.  On  potato  there  is  a 
slight  grayish  brown  growth  after  several  days,  and  subsequently, 
the  brownish  tinge  discolors  the  potato  itself.  It  forms  no  acid 
or  gas  on  any  of  the  sugars,  but  slightly  reduces  the  hydrogen  ion 
concentration  of  broth  cultures.  It  produces  ammonia  from  amino 
acids  such  as  asparagin.  It  does  not  liquefy  gelatin. 

It  is  easily  distinguished  from  B.  bronchisepticus  by  its  lack  of 
motility,  and  less  rapid  and  abundant  growth  on  artificial  media,  as 
well  as  by  agglutination  reactions.  Evans  states  from  her  studies 
that  B.  melitensis  is  closely  related  to  B.  abortus  and  can  be  dis- 
tinguished from  it  only  by  means  of  agglutination  tests.  Fleischner, 

10  Bang.  Zeit.  f.  Tier  Med.,  1,  1897,  241. 

11  Evans,  Jour.  Infec.  Dis.,  22,  1918,  580. 


800  PATHOGENIC   MICROORGANISMS 

Meyer  and  Shaw12  have  recently  examined  skin  reactions  in  connec- 
tion with  B.  abortus  bovis  and  B.  melitensis,  and  claim  that  this 
method  establishes  a  very  close  association  between  the  two. 

Of  great  importance  to  the  sanitarian  is  the  fact  that  the  B. 
abortus  may  appear  in  the  milk  of  cattle  that  have  aborted  and 
may  also  be  present  in  the  milk  of  cattle  that  are  carriers  and  have 
suffered  no  abortion  themselves.  Cotton13  has  shown  that  the  Bacil- 
lus may  persist  in  the  genital  organs  for  forty-six  days  after  aborti9n 
has  taken  place.  From  the  studies  of  Schroeder  and  Cotton,14 
Stafseth15  and  others,  it  appears  that  the  B.  abortus  does  not  es- 
tablish itself  permanently  in  the  uterine  cavity,  and  Stafseth 's 
recent  investigations  seem  to  show  that  it  does  not  penetrate  into 
the  deeper  layers  of  the  mucous  membrane  and  remain  there  as  a 
latent  infection.  • 

Theobald  Smith16  in  1912  called  attention  to  the  tuberculosis- 
like  lesions  in  guinea  pigs  caused  by  B.  abortus,  a  subject  which 
later  was  studied  in  more  detail  by  Smith  and  Fabyan.17  Fabyan, 
as  well  as  Schroeder  demonstrated  the  presence  of  the  B.  abortus 
in  milk  by  guinea  pig  injection..  Characteristic  lesions  not  unlike 
tubercles  developed  in  such  animals  in  about  eight  to  ten  weeks,  and 
Evans  found  the  Bacillus  in  the  milk  of  six  out  of  twenty-four 
cows  that  had  not  aborted.  Huddleson18  has  recently  studied 
methods  of  isolating  B.  abortus  from  milk,  other  than  by  guinea- 
pig  injection.  He  uses  a  liver  or  spleen  infusion  agar,  developed 
by  Stafseth,  a  medium  in  the  preparation  of  which  overheating  and 
filtration  through  paper  is  avoided.  The  optimum  hydrogen  ion 
concentration  is  PH  6.6.  He  adds  to  this  medium  gentian  violet 
to  a  final  concentration  of  1 :10,000.  Ten  c.c.  of  milk  is  centrifuged 
at  2,000  revolutions  for  about  two  hours.  0.1  c.c.  of  the  sediment 
is  taken  out  with  a  capillary  pipette  and  distributed  over  the  surface 
of  a  gentian  violet  agar  plate.  These  plates  are  incubated  in  jars 
in  which  about  10  per  cent  of  the  air  has  been  displaced  "by  C02. 


12  Flcischner,  Meyer  and  Shaw,  Rep.  Hooper  Foundation,  Univ.  of  Calif.,  4,  1918. 

13  Cotton,  Amer.  Veter.  Rev.,  44,  1913,  307. 

14  Schroeder  and  Cotton,  Jour.  Amer.  Vet.  Assoc.,  3,  1916,  Quoted  from  Stafseth  (3). 

15  Stafseth,  Stud,  of  Infectious  Abortion,  Mich.  Agric.  College,  49,  1920. 

16  Smith,  Theobald,  Footnote  in  article  by  Cotton,  quot.  from  Fabyn,  Jour.  Med. 
Res.,  28,  85,  1913. 

17  Smith  and  Fabyan,  Cent.  f.  Bakt.  Orig.,  61,  1912,  549. 

18  Huddleson,  Mich.  Agric,  College  Exper.  Station,  49,  Nov.  1920,  25. 


THE  BACILLUS  OF  GUINEA-PIG  PNEUMONIA  801 

By  this  method  he  was  able  to  obtain  results  comparable  in  regularity 
to  guinea-pig  inoculation,  and  obtained  cultures  in  about  four  days 
as  against  eight  weeks  by  the  guinea-pig  inoculation. 

It  is  important  in  making  routine  guinea-pig  injections  of  milk 
for  the  determination  of  tuberculosis  to  remember  the  warning  of 
Theobald  Smith19  and  not  to  jump  at  conclusions  from  mere  gross 
appearance  at  autopsy  of  the  animals. 

Whether  or  not  the  presence  of  B.  abortus  in  milk  is  a  danger 
to  man  is  not  certain,  but  it  seems  possible  that  the  organism  may 
cause  diarrheal  and  perhaps  other  diseases,  and  the  milk  of  aborted 
cattle  should  not  be  used  for  some  time  after  abortion,  and  perhaps 
subjected  to  bacteriological  test. 

The  striking  similarity  described  by  Evans  and  others  of  the 
B.  abortus  of  cattle  and  the  B.  melitensis,  taken  together  with  the 
fact  recently  observed  that  other  animals,  especially  the  goat,  may 
be  infected  by  the  B.  melitensis,  suggests  the  possibility  of  human 
pathogenicity  for  B.  abortus,  though  at  the  present  time  this  can 
be  mentioned  merely  as  a  conjecture  and  a  subject  for  inquiry. 

Bacillus  of  Guinea-Pig  Pneumonia. — In  1914  Theobald  Smith20 
called  attention  to  a  minute  motile  bacillus  originally  described  by 
Tartakowsky  which  he  found  to  be  the  cause  of  epidemic  pneumonia 
in  guinea-pigs.  He  found  it  to  be  similar  to  the  organism  described 
by  Strade  and  Traina  as  the  B.  pneumoniae  caviarum  and  Selter's 
B.  caviae  septicus.  This  organism  he  identified  with  the  B.  bron- 
chisepticus  of  McGowan.21  He  describes  the  bacillus  of  guinea-pig 
pneumonia  as  follows : 

It  is  a  minute  rod  with  rounded  ends.  From  agar  slants  it 
measures  about  0.7  micron  in  length  and  about  0.5  micron  broad. 
Longer  rods  are  occasionally  seen.  The  organisms  stain  solidly  and 
are  Gram-negative.  They  are  rapidly  motile.  They  grow  moderately 
well  on  gelatin  at  room  temperature,  but  do  not  liquefy  the  gelatin. 
On  potato  the  bacillus  makes  a  rich  yellowish-brown  color  appearing 
within  a  week.  It  clouds  broth  within  twenty-four  tours,  making 
a  delicate  iridescent  membrane  on  the  surface  after  prolonged  growth. 
Later,  a  ropy  deposit  appears.  It  makes  no  visible  change  on  milk. 
It  does  not  produce  indol.  It  is  strictly  aerobic  and  on  sugar  media 


19  Smith,  Jour.  Exper.  Med.,  30,  4,  325,  1919. 

-°  Smith,  Theobald,  Jour.  Med.  Res.,  29,  1914,  291. 

21  McGowan,  Jour.  Path,  and  Baet.,  15,  1910,  372. 


802  PATHOGENIC   MICROORGANISMS 

does  not  produce  gas  or  acid,  but  renders  broth  slowly  alkalin. 
He  states  that  in  its  bio-chemical  aspects  it  approaches  the  pyo- 
cyaneous  group,  especially  in  regard  to  its  strict  aerobiosis  and  its 
lack  of  activity  on  sugars.  In  this  respect,  Smith  calls  attention 
to  the  fact  that  it  is  similar  to  the  bacillus  of  cattle  abortion.  Smith 
compared  the  organism  with  a  culture  of  the  B.  bronchisepticus  sent 
him  by  Torrey  which  was  supposed  to  be  the  cause  of  distemper 
in  dogs,  and  found  that  his  bacillus  agreed  with  Torrey 's  strain 
in  every  particular,  in  regard  to  morphology  and  cultural  char- 
acteristics. 

He  also  identified  his  guinea-pig  bacillus  with  an  organism 
observed  by  Mallory  in  the  ciliated  epithelium  of  the  air  tubes  in 
fatal  cases  of  whooping  cough. 

In  his  description  of  the  growth  of  his  guinea-pig  organism  on 
potato,  Smith  calls  attention  to  the  similarity  of  this  bacillus  to 
the  growth  on  potato  of  the  bacillus  of  cattle  abortion  of  Bang. 

Bacillus  Pyocyaneus. — It  is  a  matter  of  common  surgical  ex- 
perience that  many  suppurating  wounds,  especially  sinuses  of  long 
standing,  discharge  pus  which  is  of  a  bright  green  color.  The  fact 
that  this  peculiar  type  of  purulent  inflammation  is  due  to  a  specific 
chromo genie  microorganism  was  first  demonstrated  by  Gessard22  in 
1882.  The  bacillus  which  was  described  by  Gessard  has  since  become 
the  subject  of  much  careful  research  and  has  been  shown  to  hold  a 
not  unimportant  place  among  pathogenic  bacteria.23 

Morphology  and  Staining. — Bacillus  pyocyaneus  is  a  short  rod, 
usually  straight,  occasionally  slightly  curved,  measuring,  according 
to  Fliigge,  about  1  to  2  micra  in  length  by  about  0.3  of  a  micron 
in  thickness.  The  bacilli  are  thus  small  and  slender,  but  are  subject 
to  considerable  variation  from  the  measurements  given,  even  in  one 
and  the  same  culture.  "While  ordinarily  single,  the  bacilli  may  be 
arranged  end  to  end  in  short  chains  of  two  and  three.  Longer 
chains  may  exceptionally  be  formed  upon  media  which  are  especially 
unfavorable  J!or  its  growth,  such  as  very  acid  media  or  those  con- 
taining antiseptics. 

Spores  are  not  found.  The  bacilli  are  actively  motile  and  possess 
each  a  single  flagellum  placed  at  one  end. 

Bacillus  pyocyaneus  is  stained  easily  with  all  the  usual  dyes,  but 
is  decolorized  by  Gram's  method.  Irregular  staining  of  the  bacillary 

22  Gessard,  Th&se  de  Paris,  1882. 

in,  "La  maladie  pyocyanique, "  Paris,  1889. 


THE  BACILLUS  PYOCYANEUS  803 

body  is  common,  but  is  always  an  indication  of  degeneration,  and 
not  a  normal  characteristic,  as,  for  instance,  in  the  diphtheria  group. 

Cultivation. — The  pyocyaneus  bacillus  is  aerobic  and  faculta- 
tively anaerobic.  It  can  be  adapted  to  absolutely  anaerobic  environ- 
ments, but  does  not  produce  its  characteristic  pigment  without  the 
free  access  of  oxygen.  The  bacillus  grows  readily  upon  the  usual 
laboratory  media  and  is  not  very  sensitive  to  reaction,  growing 
equally  well  upon  moderately  alkaline  or  acid  media.  Development 
takes  place  at  temperatures  as  low  as  18°  to  20°  C.,  more  rapidly 
and  luxuriantly  at  37.5°  C. 

On  agar  slants,  growth  is  abundant  and  confluent,  the  surface  of 
the  agar  being  covered  by  a  moist,  grayish  or  yellowish,  glistening, 
even  layer.  The  pigment  which  begins  to  become  visible  after  about 
eighteen  hours  soon  penetrates  the  agar  itself  and  becomes  diffused 
throughout  it,  giving  the  medium  a  bright  green  fluorescent  appear- 
ance, which  grows  darker  as  the  age  of  the  culture  increases. 

In  gelatin  stabs,  growth  takes  place  much  more  rapidly  upon  the 
surface  than  in  the  depths.  A  rapid  liquefaction  of  the  gelatin  takes 
place,  causing  a  saucer-shaped  depression.  As  this  deepens,  pigment 
begins  to  form  in  the  upper  layers,  often  visible  as  a  greenish  pellicle. 

In  gelatin  plates,  the  colonies  have  a  characteristic  appearance. 
They  are  round  and  are  composed  of  a  central  dense  zone,  and  a 
peripheral,  loosely  granular  zone,  which  extends  outward  into  the 
peripheral  fluidified  area  in  a  fringe  of  fine  filaments.  When  first 
appearing,  they  are  grayish  yellow,  later  assuming  the  characteristic 
greenish  hue. 

In  broth,  growth  is  rapid  and  chiefly  at  the  surface,  forming  a 
thick  pellicle.  Below  this,  there  is  moderate  clouding.  The  pigment 
is  formed  chiefly  at  the  top.  In  old  cultures  there  is  a  heavy  floc- 
culent  precipitate.  In  fluid  media  containing  albuminous  material, 
strong  alkalinity  is  produced. 

On  potato,  growth  develops  readily  and  a  deep  brownish  pigment 
appears,  which  is  not  unlike  that  produced  by  B.  mallei  upon  the 
same  medium. 

Milk  is  coagulated  by  precipitation  of  casein  and  assumes  a  yellow- 
ish-green hue.  In  older  cultures  the  casein  may  again  be  digested 
and  liquefied. 

The  pigment  of  Bacillus  pyocyaneus  has  been  the  subject  of 
much  investigation.  It  was  shown  by  Charrin24  and  others  that 

24  Charrin,  loc.  cit. 


804  PATHOGENIC   MICROORGANISMS 

this  pigment  had  no  relation  to  the  pathogenic  properties  of  the 
bacillus.  It  is  found  in  cultures  as  a  colorless  leukobase  which 
assumes  a  green  color  on  the  addition  of  oxygen.  Conversely,  the 
typical  green  "pyocyanin/'  as  the  pigment  is  called,  may  be  de- 
colorized by  reducing  substances.  This  explains  the  fact  that  it  is 
not  found  in  cultures  sealed  from  the  air.  Pyocyanin  may  be 
extracted  from  cultures  with  chloroform  and  crystallized  out  of 
such  solution  in  the  form  of  blue  stellate  crystals.  These,  on  chemical 
analysis,  have  been  found  to  belong  to  the  group  of  aromatic  com- 
pounds, with  a  formula,  according  to  Ledderhose,25  of  C14H14N20. 

Besides  pyocyanin,  Bacillus  pyocyaneus  produces  another  pig- 
ment which  is  fluorescent  and  insoluble  in  chloroform,  but  soluble 
in  water.26  This  pigment  is  common  to  other  fluorescent  bacteria, 
and  not  peculiar  to  Bacillus  pyocyaneus.  The  reddish-brown  color 
seen  in  old  cultures27  and  supposed  by  some  writers  to  be  a  third 
pigment,  is  probably  a  derivative  from  pyocyanin  by  chemical 
change. 

Chloroform  extraction  of  pyocyanin  from  cultures  may  serve 
occasionally  to  distinguish  the  pyocyaneus  bacilli  from  other  similar 
fluorescent  bacteria.  Ernst  has  claimed  that  there  are  two  types  of 
B.  pyocyaneus,  an  a-type  which  produces  only  the  fluorescent,  water- 
soluble  pigment,  and  a  /?-type  which  produces  both  this  and  pyo- 
cyanin.28 

Pathogenicity. — Bacillus  pyocyaneus  is  one  of  the  less  virulent 
pathogenic  bacteria.  It  is  widely  distributed  in  nature  and  may  be 
found  frequently  as  a  harmless  parasite  upon  the  skin  or  in  the 
upper  respiratory  tracts  of  animals  and  men.  It  has,  however,  occa- 
sionally been  found  in  connection  with  suppurative  lesions  of  various 
parts  of  the  body,  often  as  a  mere  secondary  invader  in  the  wake 
of  another  incitant,  or  even  as  the  primary  cause  of  the  inflam- 
mation. In  most  cases  where  true  pyocyaneus  infection  has  taken 
place,  the  subject  is  usually  one  whose  general  condition  and  resist- 
ance are  abnormally  low.29  Thus  pyocyaneus  may  be  the  cause  of 
chronic  otitis  media  in  ill-nourished  children.  It  has  been  cultivated 


25  Ledderhose,  quoted  from  Boland,  Cent.  f.  Bakt.,  xxv,  1889. 

26  Boland,  loc.  cit. 

27  Gessard,  Ann.  de  1'inst.  Pasteur,  1890,  1891,  and  1892. 

28  Ernst,  Zeit.  f.  Hyg.,  ii,  1887. 
"Rohner,  Cent.  f.  Bakt.,  xi,  1892, 


THE  BACILLUS  PYOCYANEUS  805 

out  of  the  stools  of  children  suffering  from  diarrhea,  and  has  been 
found  at  autopsy  generally  distributed  throughout  the  organs  of 
children  dead  of  gastro-enteritis.30  It  has  been  cultivated  from  the 
spleen  at  autopsy  from  a  case  of  general  sepsis  following  mastoid 
operation.  The  bacillus  has  been  found,  furthermore,  during  life 
in  pericardial  exudate  and  in  pus  from  liver  abscesses.31 

Brill  and  Libman,32  as  well  as  Finkelstein,33  have  cultivated 
B.  pyocyaneus  from  the  blood  of  patients  suffering  from  general 
sepsis.  Wassermann34  showed  the  bacillus  to  have  been  the 
etiological  factor  in  an  epidemic  of  umbilical  infections  in  new-born 
children.  Similar  examples  of  B.  pyocyaneus  infection  in  human 
beings  might  be  enumerated  in  large  numbers,  and  there  is  no  good 
reason  to  doubt  that,  under  given  conditions,  fatal  infections  may 
occur.  Such  cases,  however,  are  still  to  be  regarded  as  depending 
more  upon  the  low  resistance  of  the  individual  attacked  than  upon 
the  great  pathogenicity  of  B.  pyocyaneus. 

Many  domestic  animals  are  susceptible  to  experimental  pyo- 
cyaneus infection,  chief  among  these  being  rabbits,  goats,  mice,  and 
guinea-pigs.  Guinea-pigs  are  killed  by  this  bacillus  with  especial 
ease.  Intraperitoneal  inoculation  with  a  loopful  of  a  culture  of 
average  virulence  usually  leads  to  the  death  of  a  young  guinea-pig 
within  three  or  four  days. 

Toxins  and  Immunization. — Emmerich  and  Low  have  shown  that 
filtrates  of  old  broth  cultures  of  B.  pyocyaneus  contain  a  ferment-like 
substance  which  possesses  the  power  to  destroy  some  other  bacteria, 
apparently  by  lysis.  They  have  called  this  substance  ' l  pyocyanase ' ' 
and  claim  that,  with  it,  they  have  succeeded  in  protecting  animals 
from  anthrax  infection.  During  recent  years'  pyocyanase  has  been 
employed  locally  for  the  removal  of  diphtheria  bacilli  from  the 
throats  of  convalescent  cases.  Broth-culture  filtrates  evaporated  to 
one-tenth  their  volume  in  vacuo  are  used  for  this  purpose. 

Pyocyanase  is  exceedingly  thermostable,  resisting  boiling  for 
several  hours,  and  is  probably  not  identical  with  any  of  the  other 
toxins  or  peptonizing  ferments  produced  by  B.  pyocyaneus. 


80  Neumann,  Jahrb.  f.  Kinderheilk.,  1890. 

"Kraunhals,  Zeit.  f.  Chir.,  xxxvii,  1893. 

32  Brill  and  Libman,  Amer.  Jour.  Med.  Sci.,  1899. 

"Finkelstein,  Cent.  f.  Bakt.,  1899. 

84  Wassermann,  Virchow  's  Arch.,  clxv,  1901. 


806  PATHOGENIC   MICROORGANISMS 

The  toxins  proper  of  B.  pyocyaneus  have  been  the  subject  of 
much  investigation,  chiefly  by  Wasserrnann.35  Wassermann  found 
that  filtrates  of  old  cultures  were  far  more  poisonous  for  guinea-pigs 
than  extracts  made  of  dead  bacteria.  He  concludes  from  this  and 
other  observations  that  B.  pyocyaneus  produces  both  an  eudotoxin 
and  a  soluble  secreted  toxin.  The  toxin  is  comparatively  ther- 
mostable, resisting  100°  C.  for  a  short  time.  Animals  actively  im- 
munized with  living  cultures  of  B.  pyocyaneus  give  rise  in  their 
blood  serum  to  bacteriolytic  antibodies  only.  Immunized  with 
filtrates  from  old  cultures,  on  the  other  hand,  their  serum  will 
contain  both  bacteriolytic  and  antitoxic  substances.  The  true  toxin 
of  B.  pyocyaneus  never  approaches  in  strength  that  of  diphtheria 
or  of  tetanus.  Active  immunization  of  animals  must  be  done  care- 
fully if  it  is  desired  to  produce  an  immune  serum,  since  repeated 
injections  cause  great  emaciation  and  general  loss  of  strength. 
Specific  agglutinins  have  be'en  found  in  immune  sera  by  Wasser- 
mann36  and  others.  Eisenberg37  claims  that  such  agglutinins  are 
active  also  against  some  of  the  fluorescent  intestinal  bacteria. 

Bulloch  and  Hunter38  have  recently  been  able  to  show  that  old 
broth  cultures  of  B.  pyocyaneus  contain  a  substance  capable  of 
hemolyzing  the  red  blood  corpuscles  of  dogs,  rabbits,  and  sheep. 
This  "pyocyanolysin"  seems  intimately  attached  to  the  bacterial 
body.  Prolonged  heating  of  cultures  does  not  destroy  it.  Heating 
of  hemolytic  filtrates,  however,  destroys  it  in  a  short  time.  The 
filtration  of  young  cultures  yields  very  little  pyocyanolysin  in  the 
filtrate.  In  old  cultures,  however,  a  considerable  amount  passes 
into  the  filtrate.  Whether  or  not  the  hemolytic  power  is  due  to 
a  specific  bacterial  product  or  is  dependent  upon  changes  in  the 
culture  fluid,  such  as  alkalinization,  etc.,  can  not  yet  be  regarded 
as  certain. 

Gheorghiewski39  claims  to  have  found  a  leucocyte-destroying 
ferment  in  pyocyaneus  cultures. 


35  Wassermann,  Zeit.  f.  Hyg.,  xxii,  1896. 

36  Wassermann,  Zeit.  f.  Hyg.,  1902. 

37  Eisenberg,  Cent.  f.  Bakt.,  1903. 

38  Bulloch  imd  Hunter,  Cent.  f.  Bakt.,  xxviii,  1900. 

39  GheorghiewsJci,  Ann.  de  1'inst.  Pasteur,  xiii,  1899- 


CHAPTER   XLI 

PLAGUE  AND  BACILLUS  PESTIS 
(The  So-called  HaemorrJiagic  Septicaemia  Group] 

Plague. — The  history  of  epidemic  diseases  has  no  more  terrifying 
chapter  than  that  of  plague.1  Sweeping,  time  and  again,  over  large 
areas  of  the  civilized  world,  its  scope  and  mortality  were  often  so 
great  that  all  forms  of  human  activity  were  temporarily  paralyzed. 
In  the  reign  of  Justinian  almost  fifty  per  cent  of  the  entire  popula- 
tion of  the  Roman  Empire  perished  from  the  disease.  The  "  Black 
Death"  which  swept  over  Europe  during  the  fourteenth  century 
killed  about  twenty-five  million  people.  Smaller  epidemics,  appear- 
ing in  numerous  parts  of  the  world  during  the  sixteenth,  seventeenth, 
and  eighteenth  centuries,  have  claimed  innumerable  victims.  In 
1893  plague  appeared  in  Hong  Kong.  During  the  epidemic  which 
followed,  Bacillus  pestis,  now  recognized  as  the  etiological  factor 
of  the  disease,  was  discovered  by  Kitasato2  and  by  Yersin,8  inde- 
pendently of  each  other.  By  both  observers  the  bacillus  could 
invariably  be  found  in  the  pus  from  the  bubos  of  afflicted  persons. 
It  could  be  demonstrated  in  enormous  numbers  in  the  cadavers  of 
victims.  The  constancy  of  the  occurrence  of  the  bacillus  in  patients, 
shown  in  the  innumerable  researches  of  many  bacteriologists,  would 
alone  be  sufficient  evidence  of  its  etiological  relationship  to  the 
disease.  This  evidence  was  strengthened,  moreover,  by  accidental 
infections  which  occurred  in  Vienna  in  1898,  with  laboratory 
cultures. 

Since  that  time  the  investigations  of  plague  cases  and  plague 
outbreaks  by  individual  bacteriologists  and  by  commissions  of  many 
governments  have  established  the  relationship  between  the  disease 
and  the  bacillus  to  such  a  degree  that  there  is  not  the  shadow  of 
a  doubt  as  to  its  etiological  significance. 


TTaiulb.  <1.  liistor.-£ooj?r.  Path.,"  1881. 
*Kitaxato,  Lancet,  1894. 
9  Yersin,  Ann.  dc  1'inst.  Pasteur,  1894. 

807 


808  PATHOGENIC  MICROORGANISMS 

Morphology  and  Staining. — Bacillus  pestis  is  a  short,  thick 
bacillus  with  well-rounded  ends.  Its  length  is  barely  two  or  two 
and  a  half  times  its  breadth  (1.5  to  1.75  micra  by  0.5  to  0.7  micron). 
The  bacilli  appear  singly,  in  pairs,  or,  more  rarely,  in  short  chains 
of  three  or  more.  They  show  distinct  polar  staining.  In  size  and 
shape  these  bacilli  are  subject  to  a  greater  degree  of  variation  than 
are  most  other  microorganisms.  In  old  lesions  or  in  old  cultures 
the  bacilli  show  involution  forms  which  may  appear  either  as  swollen 
coccoid  forms  or  as  longer,  club-shaped,  diphtheroid  bacilli.  De- 
generating individuals  appear  often  as  swollen,  oval  vacuoles.  All 
these  involution  forms,  by  their  very  irregularity,  are  of  diagnostic 
importance.  They  appear  more  numerous  in  artificial  cultures  than 


FIG.  85. — BACILLUS  PESTIS.     (After  Mallory  and  Wright.) 

in  human  lesions.  A  very  important  property  of  the  plague  bacillus 
in  this  connection  is  the  formation  within  twenty-four  to  forty-eight 
hours  of  vacuolated  and  swollen  involution  forms  upon  salt  agar, 
that  is,  agar  to  which  3  to  5  per  cent  of  salt  is  added.  Such  a 
medium  is  of  great  value  in  diagnostic  work. 

According  to  Albrecht  and  Ghon,4  the  plague  bacillus  may,  by 
special  methods,  be  shown  to  possess  a  gelatinous  capsule.  It  does 
not  possess  flagella  and  does  not  form  spores. 

The  plague  bacillus  is  easily  stained  with  all  the  usual  anilin 
dyes.  Diluted  aqueous  fuchsin  and  methylene-blue  are  most  fre- 
quently employed.  With  these  stains  the  characteristically  deeper 

*  Albrecht  und  Ghon,  Wien,  1898. 


PLAGUE  AND  BACILLUS  PESTIS  800 

staining  of  the  polar  portions  of  the  bacillus  is  usually  easy  to 
demonstrate.  Special  polar  stains  have  been  devised  by  various 
observers.  Most  of  these  depend  upon  avoidance  of  the  usual  heat 
fixation  of  the  preparations,  which,  in  some  way,  seems  to  interfere 
with  good  polar  staining.  Fixation  of  the  dried  smears  with  absolute 
alcohol  is,  therefore,  preferable.  The  bacillus  is  decolorized  by 
Gram's  method. 

Isolation  and  Cultivation. — The  bacillus  is  easily  isolated  in  pure 
culture  from  the  specific  lesions  of  plague  patients,  during  life  or  at 
autopsy.  It  is  worth  noting  that  smears  from  bubos  and  other 
plague  lesions  will  often  show  the  typical  bacilli  in  very  small 


\ 

««> 


FIG.  86. — BACILLUS  PESTIS,  'INVOLUTION  FORMS.     (After  Zettnow.) 

numbers  only,  possibly  because  of  the  ease  with  which  they 
undergo  degeneration.  The  bacillus  grows  readily  and  luxuriantly 
upon  the  meat-infusion  media.  The  optimum  temperature  for  its 
cultivation  is  about  30°  C.  Below  20°  C.  and  above  38°  C.,  growth 
is  sparse  and  delayed,  though  it  is  not  entirely  inhibited  until 
exposed  to  temperatures  below  12°  C.,  or  above  40°  C.  The  most 
favorable  reaction  of  culture  media  is  neutrality  or  moderate  al- 
kalinity, though  slight  acidity  does  not  prevent  development. 

On  agar,  growth  appears  within  twenty-four  hours  as  minute 
colonies  with  a  compact  small  center  surrounded  by  a  broad, 
irregularly  indented,  granular  margin. 


810  PATHOGENIC   MICROORGANISMS 

On  gcMin,  similar  colonies  appear  after  two  or  three  days  at  20° 
to  22°  C.  The  gelatin  is  not  liquefied. 

In  bouillon,  the  plague  bacilli  grow  slowly.  They  usually  sink  to 
the  bottom  or  adhere  to  the  walls  of  the  tube  as  a  granular  deposit 
and  may  occasionally  form  a  delicate  pellicle.  Chain-formation  is 
not  uncommon.  In  broth  cultures,  moreover,  a  peculiar  stalactite- 
like  growth  is  often  seen,  when  the  culture  fluid  is  covered  with 
a  layer  of  oil  and  the  flasks  are  incubated  in  a  place  where  shaking 
or  vibration  can  be  prevented.  Delicate  threads  of  growth  hang 
down  from  the  surface  of  the  medium  into  its  depths  like  stalactites. 

Characteristic  involution  forms  are  brought  out  best  when  the 
.bacilli  are  grown  upon  agar  containing  3  to  5  per  cent  NaCl. 

Milk  is  not  coagulated.  In  litmus-milk  there  is  slight  acid  forma- 
tion. On  potato  and  on  blood  serum  the  growth  shows  nothing  char- 
acteristic or  of  differential  value.  On  pepton  media  no  indol  is  formed. 

Biological  Considerations. — Bacillus  pestis  is  aerobic.  Absence 
of  free  oxygen  is  said  to  prevent  its  growth,  at  least  under  certain 
conditions  of  artificial  cultivation.  It  is  non-motile.  Outside  of  the 
animal  body  the  bacilli  may  retain  viability  for  months  and  even 
years  if  preserved  in  the  dark  and  in  a  moist  environment.  In 
cadavers  they  may  live  for  weeks  and  months  if  protected  from 
dryness.  In  pus  or  sputum  from  patients  they  may  live  eight  to 
fourteen  days.  These  facts  are  of  great  hygienic  importance. 

Complete  drying  in  the  air  kills  the  bacilli  within  two  or  three 
days.5  Thoroughly  dried  by  artificial  means  they  die  within  four 
or  five  hours. 

Dry  heat  at  100°  C.  kills  the  bacillus  in  one  hour.6  Live  steam 
or  boiling  water  is  effectual  in  a  few  minutes.  The  bacilli  possess 
great  resistance  against  cold,  surviving  a  temperature  of  0°  C.  for 
as  many  as  forty  days. 

Direct  sunlight  destroys  them  within  four  or  five  hours.  The 
common  disinfectants  are  effectual  in  the  following  strengths:  car- 
bolic acid,  one  per  cent  kills  them  in  two  hours,  five  per  cent  in 
ten  minutes ;  bichloride  of  mercury  1 :1,000  is  effectual  in  ten 
minutes. 

In  a  recent  communication  to  the  New  York  Pathological  Society, 
Dr.  Wilson  reported  that  plague  cultures  which  he  had  kept  sealed 


°Kitasato,  Lancet,  1894. 

•  Abel,  Cent.  f.  Bakt..  xxi.  1897. 


PLAGUE  AND  BACILLUS  PESTIS  811 

for  as  long  as  ten  years  in  the  ice  chest  were  found  living  and 
virulent  at  the  end  of  this  time. 

In  regard  to  the  viability  of  plague  bacilli  in  air  at  different 
atmospheric  temperatures  and  conditions  of  humidity,  there  are 
many  important  sanitary  problems  involved  which  are  of  particular 
significance  in  connection  with  the  spread  of  pneumonic  plague. 
Teague  and  Barber7  worked  on  this  subject  in  connection  with  the 
Manchurian  epidemic  of  pneumonic  plague,  and  found  that  plague 
bacilli  contained  in  fine  droplets  of  pneumonic  plague  sputum  would 
suffer  death  from  drying  in  a  few  minutes  unless  they  were  sus- 
pended in  an  atmosphere  with  a  very  small  water  deficit;  in  other 
words,  the  humidity  or  the  degree  of  saturation  of  the  atmosphere 
with  water  is  a  very  important  factor  in  determining  the  length  of 
time  for  which  plague  bacilli  will  remain  alive  in  such  droplet- 
spray.  Such  atmospheres  under  ordinary  circumstances  are  common 
in  cold  climates  and  droplets  of  sputum  will,  therefore,  remain 
infectious  longer  in  cold,  wet  climates  than  in  warm  ones. 

Animal  Pathog"enicity. — Bacillus  pestis  is  extremely  pathogenic 
for  rats,  mice,  guinea-pigs,  rabbits,  and  monkeys.  The  most  sus- 
ceptible of  these  animals  are  rats  and  guinea-pigs,  in  whom  mere 
rubbing  of  plague  bacilli  into  the  unbroken  skin  will  often  produce 
the  disease.  This  method  of  experimental  infection  of  guinea-pigs 
is  of  great  service  in  isolating  the  plague  bacillus  from  material 
contaminated  with  other  microorganisms.  For  the  same  purpose, 
infection  of  rats  subcutaneously  at  the  root  of  the  tail  may  be 
employed.  Such  inoculation  in  rats  is  invariably  fatal. 

The  studies  of  McCoy8  of  the  United  States  and  Public  Health 
Service  upon  guinea-pigs  and  white  rats  show  that  individual  plague 
cultures  may  vary  considerably  in  virulence.  The  size  of  the  dose, 
always  excepting  enormous  quantities  such  as  a  whole  agar  culture, 
seems  to  make  little  difference  in  the  speed  with  which  the  animals 
die.  There  may  be  considerable  variation  in  the  susceptibility  of 
individual  animals.  Prolonged  cultivation  on  artificial  media  may 
gradually  reduce  the  virulence  of  plague  bacilli,  though,  as  stated 
above,  this  has  not  been  the  experience  of  all  observers. 


T  Teague  and  Barber,  Philippine  Jour,  of  Science,  B,  7,  1912. 

8  McCoy  noted  the  surprising  fact  that,  in  San  Francisco  a  considerable  per- 
centage of  wild  rats — especially  old  ones,  showed  a  high  natural  immunity  to 
plague. 


812  PATHOGENIC   MICROORGANISMS 

In  rats,  spontaneous  infection  with  plague  is  common  and  plays 
an  important  role  in  the  spread  of  the  disease.  The  pneumonic 
type  of  the  disease  is  common  in  these  animals  and  has  been  pro- 
duced in  them  by  inhalation  experiments.  During  every  well- 
observed  plague  epidemic,  marked  mortality  among  the  domestic 
rats  has  been  noticed. 

Although  it  was  formerly  supposed  that  rat  infection  took  place 
because  of  the  gnawing  of  dead  cadavers  by  other  rats,  the  work 
of  the  British  Indian  Plague  Commission  has  shown  that  rats,  like 
man,  are  spontaneously  infected  by  means  of  fleas  which  pass  from 
the  infected  to  the  uninfected  animal. 

In  his  work  in  California  McCoy  showed  that  the  weasel  and 
chipmunk  are  susceptible  to  plague  infection,  and  therefore,  poten- 
tial means  of  spread  if  once  infected. 

Toxin,  Formation. — The  systemic  symptoms  of  plague  are  largely 
due  to  the  absorption  of  poisonous  products  of  the  bacteria.  Al- 
brecht  and  Ghon,9  Wernicke,10  and  others  were  unable  to  obtain 
any  toxic  action  with  broth-culture  filtrates  and  concluded  that  the 
poisons  of  B.  pestis  were  chiefly  endotoxins,  firmly  attached  to  the 
bacterial  body.  Kossel  and  Overbeck,11  however,  on  the  basis,  of  a 
careful  investigation,  came  to  the  conclusion  that,  in  addition  to 
the  endotoxin,  there  is  formed  in  older  broth  cultures  a  definite  and 
important  true,  soluble  toxin. 

This,  however,  is  unlikely  in  the  light  of  a  general  survey  of 
experimental  work  and  conditions  as  they  exist  in  the  disease  itself. 
It  is  most  likely  that  the  toxic  symptoms  here  are  those  generally 
spoken  of  as  endotoxin  and  also,  we  believe,  perhaps  some  of  the 
proteose  substances  suggested  by  us  in  connection  with  other 
bacteria. 

Immunization. — A  single  attack  of  plague  usually  protects  human 
beings  from  reinfection.  A  second  attack  in  the  same  individual  is 
extremely  rare.  Immunization  in  animals  produces  specific  agglu- 
tinating and  bacteriolytic  substances  which  are  of  great  importance 
in  the  bacteriological  diagnosis  of  the  bacillus.  The  agglutinating 
action  of  the  serum  of  patients  is  clinically  important  in  the  diagnosis 
of  the  disease,  even  in  dilutions  of  one  in  ten,  since  undiluted  normal 
human  serum  has  no  agglutinating  effect  upon  plague  bacilli. 

8  Albrecht  und  Ghon,  loc.  cit. 

10  Wernicke,  Cent.  f.  Bakt.,  Ref .,  xxiv,  1898. 

11  Kossel  und  Overbeck,  Arb.  a.  d.  Gesundh.,  xviii,  1901. 


PLAGUE  AND  BACILLUS  PESTIS  813 

The  curative  plague  serum  prepared  by  Yersin  and  others  by 
the  immunization  of  horses  with  plague  cultures  has  been  extensively 
used  in  practice  and  though  often  disappointing,  a  definitely  benefi- 
cial influence  on  the  milder  cases  has  been  noted.  The  sera  are 
standardized  by  their  protective  power  as  measured  in  white  rats. 

The  question  of  prophylactic  vaccination  and  active  immuniza- 
tion will  be  taken  up  in  connection  with  plague  prevention  below. 

Plague  in  Man. — There  are  two  chief  methods  by  which  the 
disease  is  acquired  by  man.  The  first  is  by  entrance  of  the  bacilli 
through  the  skin  as  a  consequence  of  the  bite  of  an  infected  flea. 
During  the  act  of  biting,  the  flea  may  either  regurgitate  blood,  or, 
as  is  usually  the  case,  deposit  feces  on  the  skin.  The  possibilities  of 
entrance  of  the  plague  bacillus  through  minor  injuries  in  the  skin 
are  so  great  that  perhaps  the  infection  may  take  place  through  the 
lesion  caused  by  the  fleabite,  but  more  likely  is  rubbed  in  by  the 
clothing  or  by  scratching  as  the  fleabite  becomes  inflamed  and 
irritated. 

The  other  method  by  which  plague  is  transmitted  to  man  is  by 
direct  inhalation  of  sputum  spray,  a  mode  of  infection  which  causes 
pneumonic  plague.  According  to  Castellani  and  Chalmers12  and 
others,  about  2.5  per  cent  of  the  cases  occurring  during  epidemics 
of  bubonic  plague  are  of  the  pneumonic  variety,  and  there  may  be 
special  epidemics  of  pneumonic  plague  like  the  one  described  in 
another  section  and  studied  by  Strong,  Teague  and  others13  in 
Manchuria  and  a  more  recent  one  which  occurred  in  Northern  China. 

The  incubation  time  of  the  disease  is  usually  less  than  ten  days, 
and  may  be  no  longer  than  two  or  three.  The  organisms  entering 
through  the  skin  may  cause  a  localized  lesion  at  the  point  of 
entrance.  This  may  be  of  negligible  size  or  may  show  a  considerable 
inflammatory  reaction.  The  organisms  enter  the  lymphatics  and 
cause  the  so-called  bubo.  The  primary  bubos  are  situated  in  the 
glands  into  which  the  infected  area  drains  and,  for  this  reason, 
the  most  common  seat  for  these  lesions  is  in  the  glands  of  the  groin, 
but  they  also  may  be  first  seen  in  the  axillary,  cervical  or  other 
glands.  Secondary  bubos  may  arise  in  other  parts  of  the  body, 
along  the  distribution  of  lymphatics,  and  the  organisms  rapidly  enter 
the  blood  stream,  causing  septicemia. 

12  Castellani   and   Chalmers,   Manual   of   Tropical   Medicine,   W.   Wood   &    Co., 
N.  Y.,   1919. 

13  Teague  and  Strong,  Philippine  Jour,  of  Science,  Sec.  B,  No.  7,  1912. 


814  PATHOGENIC   MICROORGANISMS 

The  onset  is  usually  sudden,  with  high  fever  and  the  general 
symptoms  of  a  severe  toxemia.  Castellani  states  the  bacilli  can 
be  found  in  blood  cultures  in  about  30  per  cent  of  the  cases. 

The  disease  may  take  a  considerable  number  of  forms  which 
depend  very  largely  upon  the  virulence  with  which  the  organisms 
overwhelm  the  body.  It  may  be  relatively  mild,  or  may  take  an 
acute  septicemic  form  which  is  rapidly  fatal. 

The  pneumonic  type  is  very  severe  and  apt  to  kill  rapidly.  The 
onset  of  the  pneumonic  type  according  to  Strong  and  Teague  is 
abrupt,  without  prodromal  symptoms.  There  is  often  a  chill,  head- 
ache, and  fever  which  reaches  103°  or  104°  within  a  day  of  the 
onset,  accompanied  by  a  very  rapid  pulse.  Cough  appears  within 
twenty-four  hours.  The  expectoration  soon  becomes  abundant  and 
consists  of  blood-tinged  mucus.  When  later  it  becomes  thick  and 
bright  red,  it  contains  enormous  numbers  of  plague  bacilli.  There 
are  marked  signs  of  cardiac  involvement,  and  delirium  and  coma, 
frequently  appear.  The  same  observers  state  that  plague  bacilli 
may  frequently  be  found  in  the  blood  in  such  numbers  that  simple 
microscopical  examination  suffices  for  their  detection.  They  state 
that  in  the  Manchurian  epidemic  not  a  single  case  in  which  bac- 
teriological diagnosis  was  complete,  was  known  to  have  recovered. 

The  pathology  of  the  lungs  in  this  condition  consists  of  general 
engorgement  and  edema.  There  are  hemorrhages  under  the  pleura, 
often  fresh  fibrinous  pleurisy,  and  if  a  case  lasts  long  enough  there 
may  be  pneumonic  infiltration.  The  distribution  of  the  pneumonic 
areas  may  be  cither  lobar  or  lobular.  Bacteria  are  found  in  enormous 
numbers  in  the  peribronchial  lymph  spaces  and  in  the  adjoining 
alveoli.  They  may  also  be  present  in  large  numbers  in  the  inter- 
lobular  septa  and  under  the  pleura. 

Epidemiology. — Owing  to  the  frequency  and  wide-spread  nature 
of  plague  epidemics  in  the  history  of  the  world  from  most  ancient 
times,  it  is  quite  impossible  to  more  than  very  briefly  outline  the 
epidemiology  of  the  disease.  For  fuller  treatment  of  the  epi- 
demiological  aspects  the  reader  is  referred  to  such  books  as 
Rosenau's14  Preventive  Medicine  and  Castellani  and  ChalmerV5  work 
on  tropical  medicine.  The  prevalence  of  the  disease  in  ancient  times 

™Rosenau,  Preventive  Medicine  and  Hygiene,  D.  Appleton  &  Co.,  N.  Y.  & 
London,  1921. 

15  Castellani  and  Chalmers,  Manual  of  Tropical  Medicine,  William  Wood  &  Co., 
N.  Y.,  1919. 


PLAGUE   AND   BACILLUS   PESTIS  815 

has  been  mentioned  in  the  introduction.  Through  the  Middle  Ages 
a  number  of  plague  epidemics  swept  through  Europe  and  frequently 
reached  the  commercial  ports  of  Italy,  Asia  Minor  and  other  parts 
of  Eastern  Europe  from  the  Orient.  In  India  it  has  long  been 
known  as  a  fatal  form  of  epidemic  disease  and  since  the  early  part 
of  the  nineteenth  century,  has  probably  been  endemic  there.  Cas- 
tellani  and  Chalmers15  state  that  it  was  introduced  into  China  prob- 
ably in  the  first  half  of  the  eighteenth  century  by  Mohammedans 
returning  from  Mecca  via  Burma  to  the  Province  of  Yunnan.  Here 
it  has  been  epidemic  ever  since.  In  1894  the  study  of  the  Hongkong 
epidemic  revealed  the  causative  agent  of  the  disease.  There  was 
a  very  serious  epidemic  in  1894  which  started  in  China,  spread 
throug«h  Bombay  to  other  parts  of  India,  thence  to  Madagascar, 
into  the  Malay  States,  the  Philippine  Islands,  other  islands  of  the 
Pacific,  reaching  North  and  South  America  and  Europe;  finally 
in  1900,  it  appeared  in  Cape  Town  and  on  the  British  Isles.  Clemow, 
whom  we  quote  from  Castellani  and  Chalmers,  stated  that  in  1900 
plague  was  'endemic  in  Mongolia,  Southern  China,  the  Himalayas, 
Mesopotamia,  Persia,  Uganda,  parts  of  Russia  and  Northern  Africa. 
In  Africa  the  same  author  states  that  there  are  two  endemic  areas, 
one  in  Tripoli  and  the  other  in  Uganda  from  which  occasional 
African  epidemics  take  origin. 

The  disease  is,  thus,  a  constant  menace  in  many  different  parts 
of  the  world  and  must  remain  an  important  source  of  concern  to 
national  public  health  organizations.  In  the  United  States  the 
problem  is  perhaps  more  important  than  is  appreciated  by  the  people 
at  large.  In  1903  the  disease  appeared  in  California  and  for  several 
years  after  that  human  cases  occurred,  though  the  disease  never 
took  on  the  menace  of  an  epidemic.  This  was  prevented  probably 
by  the  energetic  work  of  the  United  States  Public  Health  Service 
under  Rupert  Blue,  McCoy,16  Curry,  and  Wherry17  who  instituted 
energetic  methods  of  rat  extermination,  rat  proofing  and  other  neces- 
sary sanitary  measures.  More  recently,  foci  have  appeared  in  Texas 
and  Now  Orleans  and  it  must  never  be  forgotten  that  the  conditions 
of  climate  and  in  other  respects  are  not  by  any  means  unfavor- 
able to  the  development  and  spread  of  plague  in  some  parts  of 
America. 

To  convey  an  idea  of  the  prevalence  of  plague  in  the  world  to-day 


16  McCoy,  Pub.  Health  Report.,  July,  1913,  No.  37. 

17  Wherry,  Jour.  Infec.  Dis.,  5,  1908. 


816  PATHOGENIC   MICROORGANISMS 

we  insert  a  record  of  plague  cases  reported  from  various  places  in 
1911,  which  we  take  from  Jackson's  Book  on  Plague.18  This  record 
appeared  in  the  British  Medical  Journal  for  September  16th,  1911 : 

India. — Deaths  from  plague  in  India  during  the  first  six  months  (of  1011), 
604,634.  Most  prevalent  (1)  United  Provinces,  281,317  (2)  Punjab,  171,084; 
(3)  Bengal,  58,515;  (4)  Bombay  Presidency,  28,109.  Deaths  in  July,  not 
included  above,  8,990. 

Hong  Kong. — April  24  to  August  21  (1911),  255  cases,  194  deaths. 

China. — Since  January  1,  1911,  plague  was  reported  in  varying  intensity 
in  (provinces  and  towns)  Manchuria,  Peking,  Tientsin,  Chefoo,  Shantung, 
Shanghai,  Amoy,  Foochow,  Swatow,  Canton,  Pakhoi  and  Laichow. 

Indo-China. — At  Saigon,  in  March  and  April  (1911),  many  cases  reported. 
April  17  to  May  7,  56  cases;  17  deaths.  May  22  to  May  28,  37  cases; 
12  deaths. 

Siam. — In  Bangkok  plague  was  more  severe  during  1911  than  in  any 
previous  year.  March  15  to  April  15,  33  cases  and  29  deaths. 

Java  and  Sumatra. — In  Java,  May  25  to  June  3  (1911),  105  cases  and 
62  deaths  (one  province).  In  Sumatra  plague  was  present,  no  statistics. 

Straits  Settlements. — A  few  cases  mostly  imported,  reported  in  1911. 

Japan. — A  few  cases  at  Kobe  in  1911.  In  Formosa,  from  April  2  to 
April  15,  31  cases ;  24  deaths. 

Egypt. — Plague  reported  from  Port  Said,  Suakin  (on  board  ship),  Cairo 
and  Alexandria ;  also  from  11  provinces.  The  province  of  Kena  had  a  severe 
outbreak,  May  5  to  May  31  (1911),  51  cases  and  49  deaths. 

Persia. — Several  cases  reported  from  ports  on  the  Persian  Gulf. 

Turkey  in  Asia. — A  few  cases  at  Muscat,  Basra  and  at  Port  of  Jeddah. 

British  East  Africa. — Kismayu  and  Port  Florence  reported  a  few  cases 
in  April  (1911). 

Mauritius. — January  1  to  April  11  (1911),  110  cases  and  70  deaths. 

Portuguese  East  Africa. — Plague  was  reported  present  at  Nahoria  in  May 
(1911). 

Eussia. — In  the  Kirgis  Steppe  in  the  Astrakan  Government  in  January 
(1911),  50  cases;  30  deaths. 

South  America. — Plague  prevailed  during  1911  in  Peru,  Ecuador,  Brazil, 
Chile  and  Venezuela.  No  severe  outbreak  except  in  Peru,  where  from 
February  to  May  many  cases  occurred  and  died.  At  Libertad,  in  March,  60 
cases  and  23  deaths  were  reported. 

Plague  is  primarily  a  disease  of  rodents.  The  bacillus  is 
pathogenic  for  rats,  mice,  guinea-pigs,  rabbits,  for  the  California 


18  Jackson,  Plague,  Lippincott  &  Co.,  1916. 


PLAUGE  AND  BACILLUS   PESTIS  817 

ground  squirrel,19  and  for  various  species  of  ground  moles  such  as 
the  Manchurian  tarbagan  (Arctomys  bobac).20 

The  spread  of  plague  by  rats  has  long  been  recognized  and  even 
in  ancient  times  mortality  in  rats  has  been  associated  with  large 
epidemic  outbreaks.  The  most  important  recent  experimental  work 
on  this  matter  was  done  by  the  British  Indian  Plague  Commission 
at  Bombay.  This  commission  demonstrated  the  relationship  between 
rats  and  plague  infection  in  carefully  conducted  experiments  in 
which  considerable  numbers  of  rats  were  used.  According  to  the 
Commission,  the  most  important  species  of  rats  are  the  Epimys  nor- 
vegicus  and  Epimys  rattus.  Over  thirteen  hundred  of  some  seventeen 
hundred  rats  found  infected,  belonged  to  these  two  species.  Other 
rats  can  also  be  infected  and  the  danger  of  plague  exists  wherever 
rats  are  found. 

The  rat  problem  is  a  very  important  one,  not  only  in  connection 
with  plague,  but  in  connection  with  economic  loss  as  well.  Creel  of 
the  United  States  Public  Health  Service21  has  called  attention  to  the 
necessity  of  rat  extermination  for  economic  reasons  alone.  The  dis- 
tribution and  number  of  rats  in  the  world  is  much  greater  than 
anyone  ordinarily  supposes.  Creel  states  that  in  the  cane  producing 
tropical  and  semi-tropical  countries,  Porto  Rico,  the  West  Indies, 
the  Hawaiian  Islands  and  the  Philippines,  there  is  an  enormous  rat 
population.  He  states  that  on  one  cane  plantation  in  Porto  Rico 
where  there  were  less  than  500  people,  25,000  rats  were  killed  in  six 
months.  He  estimates  that  in  the  United  States  the  rat  population 
is  probably  as  great  as  the  human  population,  and  the  annual  economic 
cost  per  rodent  is  higher  than  $1.00  a  piece.  Computing  the  upkeep 
of  rats  as  one-half  cent  per  day  and  estimating  their  number  as  above, 
Creel  says  that  a  sum  of  $167,000,000  is  lost  annually  to  the  country 
by  rat  depredations. 

According  to  the  British  Plague  Commission,  the  usual  way  by 
which  rats  are  infected  from  others  is  by  means  of  fleas,  and  this, 
as  first  suggested  in  1898  by  Simond,  is  the  method  by  which  the 
disease  is  carried  to  man.  In  the  British  Indian  Plague  Commission 
experiments,  when  healthy  and  infected  rats,  entirely  free  from  fleas 
were  placed  together,  no  plague  developed,  even  when  these  rats  were  in 
contact  with  the  urine  and  feces  of  the  infected  ones  and  with  polluted 
food.  But  when  fleas  were  introduced,  infection  occurred.  The  most 

19  McCoy,  Jour.  Infec.  Dis.,  5,  1909. 

20  Wu  Lien  Teh.,  Jour.  Hygiene,  13,  1913. 

n  Creel,  Rep.  No.  135,  TT.  S.  Pub.  Health  Serv.,  Vol.  28,  No.  27,  1913. 


818  PATHOGENIC   MICROORGANISMS 

common  flea  found  on  rats  is  the  Xenopsylla  cheopis.  The  disease 
can  also  be  transmitted  by  Ceratophyllu-s  fasciatus  and  by  Puiex 
irritant.  Fleas  habitually  infesting  dogs  and  cats  may  also  infest 
rats  which  means  that  flea  extermination  must  be  general.  It  also 
indicates  that  the  climatic  and  geographical  distribution  of  fleas, 
as  well  as  that  of  rats,  must  be  taken  into  account  in  dealing  with 
the  disease. 

In  rats  the  first  development  is  a  generalized  blood  infection 
during  which  enormous  numbers  of  bacilli  may  be  present  in  the 
blood.  These  are  then  taken  into  the  intestine  of  the  flea  where 
they  can  live  for  a  long  time,  and  may  be  deposted  upon  the  skin 
of  the  victim  during  feeding,  since  the  flea  is  apt  to  regurgitate 
blood  and  to  deposit  feces  at  this  time.  It  may  also  be  that  some 
of  the  bacilli  are  directly  introduced  with  the  bite,  but  it  is  probably 
more  common  that  the  organisms  thus  distributed  will  be  rubbed  in 
either  by  the  clothing  or  iri  scratching  the  fleabite. 

It  is  thus  established  with  considerable  certainty  that  while 
contact  infection  and  other  means  of  direct  and  indirect  transmis- 
sion may,  of  course,  occur,  the  usual  manner  of  spread  of  plague 
is  from  rat  to  rat,  rat  to  man,  or  man  to  man,  by  the  agency  of  fleas. 
It  is  the  Epimys  rattus  which  lives  in  closest  relationship  to  man, 
and  is  perhaps  the  most  dangerous  for  this  reason.  The  ordinary 
rat  flea  leaves  the  body  of  the  rat  within  about  three  days  of  its 
death  and  is  capable  of  remaining  alive  about  three  or  four  weeks. 
The  plague  bacilli  may  multiply  tremendously  in  the  intestine  of 
the  flea  during  the  period  between  feedings.  In  the  California 
outbreak  infection  from  ground  squirrels  to  man  was  definitely 
shown  in  a  number  of  cases,  and  in  Manchuria  the  tarbagan  men- 
tioned above  has  also  been  suspected  of  being  the  direct  source. 

McCoy22  in  1921,  summarizing  the  results  of  recent  plague  studies, 
states  that  in  the  United  States  natural  infection  has  taken  place 
among  ground  squirrels  of  California,  the  black  rats  of  Hawaii, 
and  a  species  of  wood  rat  and  field  rodent  in  Louisiana.  Human 
cases  have  been  unquestionably  traced  to  ground  squirrels,  and 
almost  always,  he  says,  have  the  peculiarity  of  showing  the  primary 
bubos  in  the  axillae,  because  the  fleas  in  the  course  of  the  infection, 
attack  the  upper  extremities,  whereas  when  the  disease  is  contracted 
from  rats,  the  fleas  are  more  apt  to  bite  on  the  legs.  Squirrel 

22  McCoy,  Amer.  Jour.  Hyg.,  March,  1921. 


PLAGUE  AND  BACILLUS   PESTIS  819 

infection,  however,  according  to  McCoy,  form  very  few  cases,  not  more 
than  about  seventeen  in  all  having  been  found  since  the  squirrel  origin 
was  first  studied.  The  squirrel  flea  can  carry  plague  from  squirrel 
to  squirrel  and  from  squirrel  to  other  rodents. 

Such  transmission  does  not  hold  good,  however,  for  the  pneumonic 
form  of  the  disease.  Careful  studies  have  been  made  on  the  pneumonic 
form  by  Strong,  Teague,  Crowell  and  Barber23  who  observed  the 
Manchurian  epidemic  which  occurred  during  the  winter  of  1910  to 
1911,  and  during  which,  within  three  months,  50,000  people  died 
of  the  disease.  According  to  these  writers  the  infection  here  is  not 
as  formerly  supposed  primarily  a  septicemic  condition,  during 
which  the  lungs  become  secondarily  involved,  but  occurs  by  direct 
inhalation  into  the  bronchi.  The  organisms  either  pass  along  the 
bronchioles  into  the  alveoli,  or  through  the  walls  of  the  bronchioles 
into  the  lungs,  giving  rise  first  to  peribronchial  inflammations  and 
later  to  more  diffuse  processes,  followed  by  pneumonic  changes  of 
the  lobar  or  lobular  type.  After  this,  the  blood  becomes  quickly 
infected  and  bacteriemia  is,  therefore,  secondary  to  pneumonia. 
As  mentioned  above,  the  organisms  are  coughed  out  with  the  drop- 
lets of  sputum,  and  thus  sprayed  into  the  atmosphere.  If  the 
atmosphere  is  dry,  they  will  rapidly  die  out.  If,  however,  the 
weather  is  cold  and  the  atmosphere  charged  with  moisture  the 
organisms  may  remain  alive  for  considerable  periods  and  inhalation 
of  virulent  organisms  may  take  place  easily.  Acording  to  the  same 
writers,  the  organisms  are  not  usually  exhaled  by  the  expired  air 
during  ordinary  respiration  or  even  during  the  labored  respirations 
of  the  pneumonic  case,  but  only  during  coughs  when  they  may 
be  sprayed  out  in  enormous  numbers  even  when  the  naked  eye 
can  detect  no  visible  spray.  In  this  form  of  plague,  then,  the 
transmission  is  very  largely  direct. 

McCoy  states  that  pneumonic  plague  rarely  occurs  from  rat 
infection,  and  states  that  it  is  an  interesting  and  perhaps  "significant 
fact"  that  in  plague  squirrels  there  seems  to  be  a  definite  tendency 
to  localize  in  the  lungs,  a  thing  which  rarely  happens  in  rats.  From 
a  study  of  the  plague  cases  in  the  United  States,  he  states  that 
except  for  one  single  focus  of  thirteen  cases,  this  form  of  the  disease 
has  not  occurred.  This  pneumonic  outbreak  originated  from  a 
bubonic  case  of  squirrel  origin  which  developed  secondary  pneu- 

23  Strong,  Teague,  Crowell  and  Barber,  Philippine  Jour.  Science,  Sec.  B,  7,  1912. 


820  PATHOGENIC   MICROORGANISMS 

monia  and  spread  through  four  transmission  generations  in  man  in 
the  autumn  of  1919. 

Plague  Prevention. — From  what  has  been  said  in  regard  to  the 
transmission  of  the  disease  it  is  apparent  that  the  prevention  of  plague 
becomes  very  largely  a  question  of  rat  extermination  and  protection 
against  fleas.  Vigilance  in  observation  of  the  mortality  among  rats  in 
endemic  centers,  for  the  discovery  of  early  rodent  foci  is  important. 
International  precautions  depend  upon  quarantine  against  rats  which 
may  easily  be  carried,  and  have  been  carried  from  country  to 
country,  by  ships  and  by  rail.  The  disinfestation  of  ships  by  S02 
by  means  of  the  Clayton  apparatus,  and  by  hydrocyanic  acid  gas 
as  described  by  Creel  and  Faget  of  the  United  States  Public  Health 
Service,  are  among  the  important  methods  in  use  for  the  disinfesta- 
tion of  ships,  sleeping  cars,  etc.  Quarantine  regulations  and  the 
supervision  of  incoming  ships  is  important.  In  the  United  States 
a  quarantine  of  seven  days  is  imposed  on  ships  arriving  from  plague 
ports,  a  period  which  is  probably  not  long  enough.  Precautions 
must  be  taken  to  prevent  the  travel  of  rats  along  hawsers  when 
ships  are  docked  at  a  wharf,  and  this  is  usually  accomplished  by 
the  application  of  large  circular  shields  along  the  course  of  the 
hawsers  in  such  a  way  that  rats  cannot  cross. 

When  foci  of  plague  are  discovered  in  any  community,  wholesale 
rat  destruction  and  isolation  of  the  focus,  by  destruction  of  build- 
ings, ratp roofing  of  cellars,  etc.,  must  be  resorted  to.  Blake  has 
introduced  a  system  of  which  Castellani  and  Chalmers  speak  very 
highly,  the  principle  of  which  is  that  the  rat  extermination  and 
other  precautionary  measures  are  started  in  a  wide  circle  about 
the  focus,  working  in  toward  the  center,  since  work  beginning  at 
the  focus  itself  in  an  outward  circle  may  easily  serve  to  scatter 
rats,  rather  than  circumscribe  them.  In  the  Philippines  and  in 
villages  in  which  natives  live  in  primitive  huts,  actual  burning  of 
the  houses  has  been  resorted  to,  but  this,  too,  may  easily  result 
in  merely  scattering  the  rat  population  into  the  neighboring  districts. 
On  a  large  scale,  rat  extermination  is  usually  carried  out  by  poisons 
in  which  phosphorous  paste  is  perhaps  the  most  important  method. 
Of  especial  importance  is  the  protection  of  food  stores,  and  particular 
attention  to  all  depositories  of  food,  grain,  etc.,  about  which  rats 
are  apt  to  accumulate. 

BACTERIOLOGICAL  DIAGNOSIS  OF  SUSPECTED  PLAGUE  CASES. — Since 
the  bacteriological  diagnosis  of  the  earliest  cases  that  occur  is  one  of 


PLAGUE  AND  BACILLUS  PESTIS  821 

the  most  important  problems  of  prevention,  various  governments 
have  laid  down  methods  of  collection  and  shipment  of  material  that 
should  be  followed  in  the  case  of  suspected  cases  in  man  and  rats. 
Public  Health  Reports,  Volume  35,  Number  37,  lays  down  the  method 
in  which  material  is  to  be  collected  for  the  United  States.  This  we 
quote  in  toto  from  this  Bulletin  as  follows: 
To  the  Officers  of  the  Public  Health  Service  and  State  and  Local  Health 

Officers  : 

Owing  to  the  appearance  of  plague  in  several  American  ports  it  is 
important  that  all  cases  of  suspected  plague,  both  in  man  and  animals,  be 
subjected  to  a  bacteriological  examination. 

1.  The  following  material  from  persons  or  rodents  suffering  from  plague 
may  be  sent  to  laboratories: 

Human  Cases   (Living) 

(a)  Pus  or  gland  fluid  from  buboes   aspirated  by  syringe  or  collected 

after  incision,  on  agar  slants. 

(b)  Portions  of  tissues  affected,  removed  at  operation,  in  sterilized  bottles, 

securely  stoppered. 

(c)  Blood  specimens,  in  sterilized  sealed  glass  ampules  or  test  tubes. 

(d)  Cultures  of  suspected  organisms,  on  agar  slants. 

Human  Cases  (Necropsy) 

(a)   Portions  of  the  affected  tissues — preferably  bubo,  lung  and  spleen — 
in  sterilized  glass  bottles,  securely  stoppered. 

Rodents 
(a)   The  whole  rodent  carcass,  in  fruit  preserving  jar. 

2.  Do  not  place  tissues  or  rodents  in  a  preservative.    The  bacteriological 
diagnosis  of  plague  rests  upon  the  production  of  the  disease  in  laboratory 
animals,  and  the  isolation  and  growth  of  the  causative  organism,  Bacillus 
pestis.     Any  preservative  that  kills  this  organism  will  defeat  the  purpose 
of  the  examination.     If  decomposition  of  the  specimen  is  feared,  it  may  be 
placed  in  a  tight  container  and  this  in  turn  surrounded  by  ice  in  a  larger 
container,  preferably  of  wood.     Every  specimen  should  be  plainly  marked 
preferably  by  ordinary  pencil,  showing  the  date  and  the  exact  location  from 
which  it  was  taken. 

3.  The  shipper  must  make  certain  that  the  specimen  is  packed  in  such 
manner  as  to  prevent  possible  danger  to  those  handling  the  same,  provided 
the  package  is  properly  handled. 

In  this  connection  it  is  necessary  that  specimens  be  wrapped  in  sufficient 
cotton  or  other  absorbent  material,  to  prevent  leakage  of  fluid  from  the 
container  should  the  glass  be  broken. 

The  Following  Instructions  should  be  explicitly  observed. 

1.  Ship  by  express — Federal  laws  prohibit  the  shipping  of  plague-infected 
material,  or  cultures,  by  mail. 


822  PATHOGENIC   MICROORGANISMS 

2.  Do  not  make  packages  too  small,  as  small  packages  are  more  likely 
to  be  lost  in  transit,  or  overlooked. 

3.  Each  package  should  be  marked  as  follows: 

Notice 

This  package  contains  perishable  specimens 

for  bacteriological  examination 

Please  Expedite 

Careful  autopsy  must  of  course  be  made  on  all  cases,  animal  or 
man,  and  the  lesions  studied.  The  lesions  in  rats  have  been  fully 
described  in  another  section.  Cultures  are  taken  on  agar  and  smears 
taken  from  buboes  or  sputum,  stained  by  Loeffler's  methylene-blue, 
the  bipolar  appearance  and  degeneration  forms  of  the  organisms 
looked  for.  Cultural  diagnosis  is  then  made  by  the  appearance 
of  the  growing  organisms,  and  their  colonies,  the  staining  properties, 
appearance  on  salt  agar,  agglutination  in  immune  sera,  and,  above 
all,  inoculation  of  rats  and  guinea-pigs  with  observation  of  the 
characteristic  lesions  in  these  animals. 

Since  the  examination  of  rats  for  plague  is  an  important  phase 
of  the  study  of  epidemics,  it  may  be  well  to  review  the  typical  lesions 
in  these  animals  as  described  by  an  experienced  American  student 
of  plague,  George  W.  McCoy.24  McCoy,  agreeing  with  the  Indian 
Plague  Commission,  states  that  the  naked  eye  is  superior  to  the 
microscopical  examination.  There  is  engorgement  of  the  subcutane- 
ous vessels  and  a  pink  coloration  of  the  muscles.  The  bubo  when 
present  is  sufficient  for  diagnosis.  Marked  injection  surrounds  it 
and  sometimes  there  is  hemorrhagic  infiltration.  The  gland  itself 
is  firm  but  usually  caseous  or  occasionally  hemorrhagic.  In  the  liver 
there  is  apparent  fatty  change,  but  this  is  due  to  necrosis.  Pin-point 
spots  give  it  a  stippled  appearance  as  though  it  had  been  dusted 
with  pepper.  Pleural  effusion  is  an  important  sign.  The  spleen 
is  large,  friable,  and  often  presents  pin-point  granules  on  the  surface. 
One  or  two  per  cent  of  rats  may  present  no  gross  lesions.  Cultures 
should  of  course  be  made.  The  method  of  examination  consists  in 
immersing  the  rat  in  any  convenient  antiseptic  to  kill  fleas  and  other 
ectoparasites.  The  rats  ar*e  nailed  by  their  feet  to  a  shingle  and 
the  skin  is  reflected  from  the  whole  front  of  the  body  and  neck, 
so  as  to  expose  the  cervical,  axillary,  and  inguinal  regions.  The 
thoracic  and  abdominal  cavities  are  then  opened  and  examined. 


24  McCoy,  Jour,  of  Inf.  Dis.,  vi,  1909 ;  George  W.  McCoy,  Public  Health  Beports, 
July,   1912. 


PLAGUE  AND   BACILLUS   PESTTS  823 

An  excellent  example  of  the  circumscription  of  a  plague  focus 
at  its  first  discovery  is  one  which  we  take  from  a  note  by  Rucker 
in  the  United  States  Public  Health  Service  Report,  No.  28,  1915, 
based  largely  on  the  work  of  Passed  Assist.  Surg.  R.  A.  Kearny. 

In  September,  1914,  a  dead  Mus  norvegicus  was  found  on  a  street  corner 
in  New  Orleans.  Laboratory  examination  proved  this  plague  infected.  The 
district  was  searched  for  other  rats  and  on  the  16th  of  September  a  similar 
plague  rat  was  found  in  the  neighborhood  in  a  Chinese  restaurant  located 
in  a  ramshackle  frame  building,  situated  between  a  rat-proof  brick  building 
and  an  open  lot.  Behind  the  restaurant  was  a  frame  shed  which  was  not 
rat  proof.  A  survey  of  the  district  followed,  in  which  thirty-eight  infected 
rats  were  discovered,  all  of  them  of  the  same  species  as  the  preceding.  One 
hundred  and  thirteen  dead  rats  were  found,  and  two  infected  rats  were  found 
on  a  neighboring  street  corner.  Twenty-one  were  found  in  the  Chinese 
restaurant,  and  one  in  the  open  lot  and  the  other  in  the  neighborhood.  Rucker 
believes  that  the  focus  was  eliminated  largely  because  there  was  plenty  of  food 
in  this  particular  neighborhood,  and  rats  could  not  easily  leave  there  without 
entering  the  street,  a  thing  which  they  would  have  done  only  under  the 
pressure  of  shortage  of  food  supply.  He  calls  attention  to  the  fact  that 
if  this  had  been  a  focus  of  Mus  ratus  or  Mus  alexandrinus  which  are  climbing 
rats,  the  original  focus  would  rapidly  have  been  spread.  But  since  the 
Mus  norvegicus  is  a  ground  rat,  it  was  closed  in  by  the  neighboring  brick 
walls.  In  the  operations  following  these  discoveries,  the  building  chiefly 
infested  was  torn  down  and  the  frame  sheds  behind  it  were  rendered  not 
inhabitable  for  rats.  Many  rats  were  found  dead  and  a  considerable  number 
were  killed.  Fumigation  was  carried  out  on  the  premises,  and  these  and 
other  premises  washed  down  with  tank  oil  for  the  purpose  of  killing  fleas. 
Very  few  rats  escaped.  An  interesting  control  was  carried  out  which  has 
been  introduced  into  plague  campaigns,  namely,  that  guinea-pigs  were  placed 
into  the  fumigated  premises  after  fumigation.  One  of  these  contracted  plague 
and  died,  and  the  place  was,  therefore,  refumigated.  Guinea-pigs  which 
had  been  used  as  controls  in  other  places  remained  alive.  Only  one  human 
case  was  attributable  to  this  focus. 

A  Bulletin  published  by  the  United  States  Public  Health  Service 
in  November,  1920,  (35,  No.  45)  has  laid  down  ordinances  for  rat- 
proofing.  These  we  quote  in  toto  directly  from  this  Bulletin. 

"The  rat-proofing  of  buildings  is  generally  secured  either  by  elevation 
of  the  structure,  with  the  underpinning  open  and  free,  or  by  marginal 
rat-proof  walls  of  concrete  or  of  stone  or  brick  laid  in  cement  mortar, 
sunk  two  feet  into  the  ground,  and  fitting  flush  to  the  floor  above.  The 
wall  must  fit  tightly  to  the  flooring  and  not  merely  extend  to  the  joists  or 


824  PATHOGENIC   MICROORGANISMS 

supporting  timbers,  as  this  would  result  in  open  spaces,  permitting  the 
entrance  of  rodents.  Groceries,  stables,  warehouses,  markets,  and  food  depots 
in  general  are  best  rat-proofed  by  having  a  concrete  floor  in  addition  to 
concrete  walls.  In  these  structures,  untenanted  as  they  are  at  night,  rats 
might  well  enter  by  a  doorway  or  window  carelessly  left  open,  or  be  intro- 
duced concealed  in  merchandise,  and,  gnawing  through  plank  flooring,  obtain 
a  well-protected  hiding  place. 

"In  addition  to  concrete  floor  and  walls,  these  food  depots  must  have 
tight-fitting  doors,  and  all  windows  and  other  openings  should  be  properly 
screened.  A  12-guage  wire  is  preferable  on  account  of  its  strength  and 
durability,  and  the  mesh  should  not  be  larger  than  one-half  inch. 

"Rat-proofing  by  elevation  of  the  building  is  chiefly  applicable  to  small 
and  medium  sized  frame  dwellings.  The  purpose  is  to  have  a  sufficient 
elevation,  about  two  feet,  so  that  the  ground  area  beneath  will  be  as  exposed 
and  free  from  covert  as  land  unbuilt  upon.  Marginal  rat-proofing  will 
suffice  in  more  pretentious  dwellings  where  sufficient  care  can  be  exercised 
to  prevent  rats  from  gnawing  through  the  plank  floors. 

"Chicken  pens  can  be  protected  by  marginal  concrete  walls,  sunk  into 
the  ground  two  feet  or  more,  and  by  covering  the  sides  and  top  with  ^-inch 
mesh  wire  netting.  Garbage  cans  should  be  made  of  serviceable  metal  and 
should  have  properly  fitting  tops. 

"Plank  sidewalks  and  plank  coverings  for  yards  should  be  avoided. 
Cinders  and  concrete  should  be  used  instead.  The  latter  should  have  marginal 
protection  to  prevent  rats  from  burrowing  beneath  it. 

"Double  walls,  with  a  dead  space  between,  should  be  avoided,  or,  if  used, 
they  should  be  rat-proofed  at  the  top  and  bottom  with  heavy  wooden  timbers, 
4  by  4-inch  fillers,  or  by  a  concrete  fill.  Attics  should  be  well  opened  and 
kept  free  of  rubbish  or  other  refuge  for  rats. 

"These  precautions  against  rat  harborage  and  for  the  protection  of  food 
supplies,  in  connection  with  careful  trapping  and  poisoning,  will  be  attended 
with  considerable  success  in  the  destruction  of  rats. 

"The  appended  model  ordinance  is  applicable,  with  perhaps  slight  modifi- 
cations, to  any  urban  community.  It  should  be  examined  by  competent  local 
counsel  for  changes  in  form,  or  in  substance  if  necessary,  as  dictated  by 
special  constitutional,  legislative,  or  charter  considerations." 

Plague  Vaccination. — The  immunization  of  animals  with  suspen- 
sions of  plague  bacilli,  killed  by  moderate  heating,  50°  for  one 
hour,  was  first  attempted  by  Yersin,  Calmette  and  Borrel  in  1897. 
Kolle,25  Haffkine26  and  others  studied  plague  vaccination  particularly 
in  the  subsequent  years.  A  great  many  different  vaccines  have  been 

x  Kolle,  loc.  cit. 
"Haffkine,  loc.  cit. 


PLAGUE  AND  BACILLUS   PESTIS  825 

introduced  since  then.  The  one  most  extensively  used  is  that  of 
Haffkine,  which  consists  of  cultures  grown  in  broth  in  shallow  bottles 
for  six  weeks  at  room  temperature  and  shaken  once  a  day.  At  the 
end  of  this  time  they  are  sterilized  at  65°  for  several  hours.  The 
material  then  consists  of  degenerated  organisms  and  extracts  of 
the  organisms.  Glycerinated  broth  cultures  have  been  introduced, 
but  so  far  have  had  little  practical  application.  The  German  Plague 
Commission27  in  1899  introduced  the  use  of  heated  cultures  to  which 
0.5  per  cent  carbolic  acid  had  been  added.  Strong28  believing  that 
attenuated  living  bacilli  might  be  more  efficient  than  dead  cultures, 
produced  vaccines  from  a  three  years'  old  laboratory  culture,  sub- 
sequently cultivated  at  temperatures  above  41°.  These  living  cul- 
tures after  such  treatment  had  lost  their  virulence  for  guinea-pigs 
and  monkeys  almost  completely.  He  vaccinated  forty-two  individ- 
uals with  these  cultures  without  harm,  and  with  resulting  develop- 
ment of  specific  antibodies.  The  method  is  probably  quite  efficient 
but  because  of  the  possible  danger  involved  in  it,  has  not  been 
extensively  employed.  The  so-called  nucleo-protein  vaccins  of  Lus- 
tig  and  Galeotti,29  that  is,  plague  bacilli  extracted  with  weak  alkalin 
solutions  and  precipitated  with  acid  in  the  cold,  have  been  studied 
extensively  by  Rowland30  and  others,  and  Rowland  made  several 
vaccines  of  his  own  in  one  of  which  he  extracted  the  bacterial  mass 
with  sodium  sulphate.  He  also  used  cultures  killed  with  chloroform. 
None  of  these  vaccines  have  had  any  extensive  application  except 
that  of  Haffkine  which  has  been  used  by  the  British  Government 
Sanitary  organization  in  India  on  a  very  large  scale.  Bannermann, 
Bitter  and  more  recently,  Major  Glen  Listen31  have  analyzed  the 
results  obtained  with  Haffkine 's  virus  of  which  over  eight  million 
doses  were  distributed  in  India  between  1886  and  1899.  According 
to  these  studies  the  Haffkine  virus  seems  to  be  of  definite  prophy- 
lactic value,  though  not  completely  protective  as  one  would  expect 
from  the  nature  and  virulence  of  the  disease.  Major  Listen  states 
in  his  report  of  the  Bombay  Bacteriological  Laboratory  for  the  years 
1913  to  1916,  that  it  is  quite  impossible  to  give  any  positive  state- 
ment for  India,  but  that  in  isolated  epidemics  in  which  careful  figures 

"German  Plague  Commission  Report,  1899. 

**  Strong,  Philip.  Jour.  Science.,  Sec.  B,  1907  and  1912. 

*Lustig  and  Galeotti,  Deut.  med.  Woch.,  1897-1912. 

80  Rowland,  Journal  of  Hygiene,  1910-1914. 

"Liston,  Maj.  Glen,  Bombay  Bacter.  Lab.  Rep.,  1913-1916. 


826  PATHOGENIC   MICROORGANISMS 

could  be  secured,  the  indications  are  that  the  vaccine  has  been  of 
great  value.  In.  one  town  in  India,  794  individuals  were  inoculated, 
and  286  uninoculated,  and  among  the  inoculated  there  were  only 
twelve  cases  and  three  deaths,  while  among  the  smaller  number 
of  the  untreated,  thirty  cases  developed  and  twenty-five  died.  In 
one  house  there  were  four  vaccinated  and  three  unvaccinated.  All 
of  the  unvaccinated  died,  and  only  one  of  the  vaccinated  contracted 
the  disease  and  he  recovered.  A  number  of  similar  studies  are  cited 
by  Major  Liston.  There  seems,  therefore,  to  be  very  little  doubt 
as  to  the  protective  value  of  some  form  of  plague  vaccination. 
Whether  or  not  the  Haffkine  virus  is  the  most  useful  and  final 
method,  cannot  of  course  be  stated  at  the  present  time. 

THE  PLAGUE-LIKE  DISEASE  OF  RODENTS  (McCOY)32 

Bacterium  Tularense   (McCoy  and  Chapin)33 

McCoy  has  described  a  disease  occurring  in  Californian  ground 
squirrels  (Citellus  beechyi)  which  presents  lesions  very  similar  to 
those  of  plague  in  these  animals.  In  fact  the  disease  was  noticed 
in  the  course  of  the  systematic  examination  of  rodents  by  McCoy 
at  the  Federal  Laboratory  in  San  Francisco.  Although  McCoy  was 
able  to  transmit  the  disease  to  guinea-pigs,  mice,  rabbits,  monkeys, 
and  gophers,  and  plague-like  lesions  could  be  produced  in  most  of 
the  animals,  he  was  at  first  entirely  unable  to  cultivate  any  organism 
from  these  lesions.  In  1912  McCoy  and  Chapin  finally  succeeded 
in  growing  the  specific  bacterium  on  an  egg  medium  made  entirely 
of  the  yolk.  Morphologically  it  is  a  very  small  rod,  0.3  to  0.7  micron 
in  length  and  often  capsulated.  The  rods  stain  poorly  with 
methylene  blue,  better  with  carbol  fuchsin  or  gentian  violet.  They 
are  found  in  large  numbers  in  the  spleen  of  animals  dead  of  the 
disease. 

THE  BACILLI  OF  THE  HEMORRHAGIC  SEPTICEMIA  GROUP 

In  many  of  the  lower  animals  there  occur  violently  acute  bacterial 
infections  characterized  by  general  septicemia,  usually  with  petechial 
hemorrhages  throughout  the  organs  and  serous  membranes  and 
severe  intestinal  inflammations.  These  diseases,  spoken  of  as  the 
"hemorrhagic  septicemias, "  are  caused  by  a  group  of  closely  allied 

82  McCoy,  U.  S.  Public  Health,  Bull.  43,  1911. 

83  McCoy  and  Chapin,  Jour,  of  Inf.  Dis.,  x,  1912. 


PLAGUE  AND  BACILLUS   PESTIS  827 

bacilli,  first  classified  together  by  Hueppe34  in  1886.  Some  confusion 
has  existed  as  to  the  forms  which  should  be  considered  within 
Hueppe 's  group  of  "hemorrhagic  septicemia,"  a  number  of  bac- 
teriologists including  in  this  class  bacilli  such  as  Loeffler's  Bacillus 
typhi  murium,  and  Salmon  and  Smith's  hog-cholera  bacillus,  micro- 
organisms which,  because  of  their  motility  and  cultural  character- 
istics, belong  more  properly  to  the  "Gartner,"  "enteritidis,"  or 
"paratyphoid"  group,  intermediate  between  colon  and  typhoid. 

The  organisms  properly  belonging  to  this  group  are  short  bacilli, 
more  plump  than  are  those  of  the  colon  type,  showing  a  marked 
tendency  to  stain  more  deeply  at  the  poles  than  at  the  center.  They 
are  non-motile,  possess  no  flagella,  and  do  not  form  spores.  They 
grow  readily  upon  simple  media,  but  show  a  very  marked  preference 
for  oxygen,  growing  but  slightly  below  the  surface  of  media.  By 
some  observers  they  are  characterized  as  "obligatory  aerobes, "  but 
this  is  undoubtedly  a  mistake. 

While  showing  considerable  variations  in  form  and  differences 
in  minor  cultural  characteristics,  the  species  characteristics  of  polar 
staining,  decolorization  by  Gram,  immobility,  lack  of  gelatin  lique- 
faction, and  great  pathogenicity  for  animals,  stamp  alike  all  mem- 
bers of  the  group.  Its  chief  recognized  representatives  are  the 
bacillus  of  chicken  cholera,  the  bacillus  of  swine-plague  (Deutsche 
Schweineseuche),  and  the  Bacillus  pleurosepticus  which  causes  an 
acute  disease  in  cattle  and  often  in  wild  game. 

Because  of  certain  cultural  and  pathogenic  characteristics,  it 
seems  best  to  consider  the  bacillus  of  bubonic  plague  with  this  group. 

BACILLUS  OP  CHICKEN  CHOLERA 

(Bacillus  avisepticus) 

The  bacillus  of  chicken  cholera  was  first  carefully  studied  by 
Pasteur35  in  1880.  It  is  a  short,  non-motile  bacillus,  measuring  from 
0.5  to  1  micron  in  length.  Stained  with  the  ordinary  anilin  dyes, 
it  displays  marked  polar  staining  qualities,  which  often  give  it  the 
appearance  of  being  a  diplococcus.  It  is  decolorized  by  Gram's 
method.  It  does  not  form  spores,  but  may  occasionally  form 
vaciiolated  degeneration  forms,  not  unlike  those  described  for  Bacil- 
lus pestis. 

34  Hueppe,  Berl.  klin.  Woch.,  1886. 

35  Pasteur,  Comptes  rend,  cle  Pacad.  des  sci.,  1880. 


828  PATHOGENIC   MICROORGANISMS 

The  bacillus  is  easily  cultivated  from  the  blood  and  organs  of 
infected  animals,  it  grows  well  upon  the  simplest  media  at  tempera- 
tures varying  from  25°  to  40°  C.  In  broth,  it  produces  uniform 
clouding  with  later  a  formation  of  a  pellicle.  Upon  a  gar  it  forms, 
within  twenty-four  to  forty-eight  hours,  minute  colonies,  white  or 
yellowish  in  color,  which  are  at  first  transparent,  later  opaque.  Upon 
gelatin,  it  grows  without  liquefaction.  Upon  milk,  the  growth  is 
slow  and  does  not  produce  coagulation.  According  to  Kruse,36  indol 
is  formed  from  pepton  bouillon.  Acid,  but  no  gas,  is  formed  in  sugar 
broth. 

Among  barnyard  fowl,  this  disease  is  widely  prevalent,  attacking 
chickens,  ducks,  geese,  and  a  large  variety  of  smaller  birds.  The 
infection  is  extremely  acute,  ending  fatally  within  a  few  days.  It 
is  accompanied  by  diarrhea,  often  with  bloody  stools,  great  exhaus- 
tion, and,  toward  the  end,  a  drowsiness  bordering  on  coma.  Autopsy 
upon  the  animals  reveals  hemorrhagic  inflammation  of  the  intestinal 
mucosa,  enlargement  of  the  liver  and  spleen,  and  often  broncho- 
pneumonia. 

The  specific  bacilli  may  be  found  in  the  blood,  in  the  organs, 
in  exudates,  if  these  are  present,  and  in  large  numbers  in  the  dejecta. 
Infection  takes  place  probably  through  the  food  and  water  con- 
taminated by  the  discharges  of  diseased  birds.37 

Subcutaneous  inoculation  or  feeding  of  such  animals  with  pure 
cultures,  even  in  minute  doses,  gives  rise  to  a  quickly  developing 
septicemia  which  is  uniformly  fatal.  The  bacillus  is  extremely 
pathogenic  for  rabbits,  less  so  for  hogs,  sheep,  and  horses,  if  infection 
is  practiced  by  subcutaneous  inoculation.  Infection  by  ingestion 
does  not  seem  to  cause  disease  in  these  animals. 

Historically,  the  bacillus  of  chicken  cholera  is  extremely  in- 
teresting, since  it  was  with  this  microorganism  that  Pasteur35  carried 
out  some  of  his  fundamental  researches  upon  immunity,  and  suc- 
ceeded in  immunizing  chickens  with  attenuated  cultures.  The  first 
attenuation  experiment  made  by  Pasteur  consisted  in  allowing  the 
bacilli  to  remain  in  a  broth  culture  for  a  prolonged  period  without 
transplantation.  With  minute  doses  of  such  a  culture  (vaccin  I) 
he  inoculated  chickens,  following  this,  after  ten  days,  with  a  small 
dose  of  a  fully  virulent  culture.  Although  enormously  important 
in  principle,  the  practical  results  from  this  method,  as  applied  to 

88  Kruse,  in  Fliigge  's  ' '  Die  Mikroorganismen. ' ' 

87  Salmon,  Rep.  of  the  Com.  of  Agriculture,  1880,  1881,  and  1882. 


PLAGUE  AND  BACILLUS   PESTIS  829 

chicken  cholera,  have  not  been  satisfactory.  It  was  with  this  bacil- 
lus, furthermore,  that  Pasteur  was  first  able  to  demonstrate  the 
existence  of  a  free  toxin  which  could  be  separated  from  the  bacteria 
by  filtration. 

BACILLUS  OF  SWINE  PLAGUE 

(Bacillus  suisepticus,  Schweineseuche) 

This  microorganism  is  almost  identical  in  form  and  cultural 
characteristics  with  the  bacillus  of  chicken  cholera.  It  is  non-motile, 
forms  no  spores,  is  Gram-negative,  and  does  not  liquefy  gelatin. 
The  bacillus  causes  an  epidemic  disease  among  hogs,  which  is  char- 
acterized almost  regularly  by  a  bronchopneumonia  followed  by 
general  septicemia.  There  is  often  a  sero-sanguineous  pleura! 
exudate,  a  swelling  of  bronchial  lymph  glands  and  of  liver  and 
spleen.  The  gastrointestinal  tract  is  rarely  affected.  The  bacilli 
at  autopsy  may  be  found  in  the  lungs,  in  the  exudates,  in  the  liver 
and  spleen,  and  in  the  blood.  The  disease  is  rarely  acute,  but,  in 
young  pigs,  almost  uniformly  fatal. 

It  is  probable  that  spontaneous  infection  usually  occurs  by  in- 
halation. Experimental  inoculation  is  successful  in  pigs,  both  when 
given  subcutaneously  and  when  administered  by  the  inhalation 
method.  Mice,  guinea-pigs,  and  rabbits  are  also  susceptible,  dying 
within  three  or  four  days  after  subcutaneous  inoculation  of  small 
doses. 

Active  and  passive  immunization  of  animals  against  Bacillus 
suisepticus  has  been  attempted  by  various  observers.  Active  im- 
munization, if  carried  out  with  care,  may  be  successfully  done  in 
the  laboratory.  Passive  immunization  of  animals  with  the  serum  of 
actively  immunized  horses  has  been  practiced  by  Kitt  and  Mayr,38 
Schrieber,39  and  Wassermann  and  Ostertag.  The  last-named  ob- 
servers, working  with  a  polyvalent  serum  produced  with  a  number 
of  different  strains  of  the  bacillus,  have  obtained  results  of  consider- 
able practical  value.  The  researches  of  Kitt  and  Mayr  have  revealed 
a  fact  pointing  to  the  interrelationship  of  the  bacilli  of  the  "hemor- 
rhagic  septicemia"  group.  They  were  able  to  show  that  the  serum 
of  horses  immunized  with  chicken  cholera  bacilli  was  able  to  protect, 
somewhat,  against  Bacillus  suisepticus. 


*Kitt  and  Mayr,  Monatsschr.  f.  Thier  Leilk.,  vol.  8,  1897. 
39  Schrieber,  Berl.  Thierarztl.  Wochenschr.,  vol.  10,  1899. 


830  PATHOGENIC   MICROORGANISMS 

Infection  with  the  bacillus  of  swine  plague,  in  hogs,  is  often 
accompanied  by  an  infection  with  the  hog-cholera  bacillus  (Schweine- 
pest).  The  latter,  as  we  have  seen,  is  a  microorganism  belonging 
to  the  enteritidis  group,  intermediate  between  Bacillus  coli  and 
Bacillus  typhosus,  and  differing  from  suisepticus  in  being  actively 
motile,  possessing  flagella,  not  showing  the  polar  staining,  having 
a  more  slender  morphology,  and  producing  gas  upon  dextrose  broth. 
A  confusion  between  the  two  bacilli  frequently  occurs  because  of 
their  nomenclature.  Bacteriologically  and  pathogenically,  they  are 
quite  distinct.  Bacillus  suisepticus  produces  an  acute  septicemia, 
accompanied  by  bronchopneumonia  and  usually  not  affecting  the 
gastro-intestinal  canal.  The  bacillus  of  hog  cholera  produces  an 
infection  localized  in  the  intestinal  canal. 


CHAPTER   XLII 

ASIATIC  CHOLERA  AND  THE  CHOLEEA  ORGANISM 
(Spirillum  choleroz  asiaticce,  Comma  Bacillus) 

THE  organism,  of  Asiatic  cholera  was  unknown  until  1883.  In 
this  year,  Koch/  at  the  head  of  a  commission  established  by  the 
German  government  to  study  the  disease  in  Egypt  and  India,  dis- 
covered the  " comma  bacillus"  in  the  defecations  of  patients,  and 
satisfactorily  determined  its  etiological  significance. 

Koch's  investigations  were  carried  out  on  a  large  number  of 
cases  and  many  investigations  have  since  then  corroborated  his 
results. 

Apart  from  the  evidence  of  the  constant  association  of  the  cholera 
spirillum  with  the  disease,  the  etiological  relationship  has  been 
clearly  demonstrated  by  several  accurately  recorded  accidental  in- 
fections occurring  in  bacteriological  workers,  and  by  the  famous 
experiment  of  Pettenkofer  and  Emmerich,  who  purposely  drank 
water  containing  cholera  spirilla.  Both  observers  became  seriously 
ill  with  typical  clinical  symptoms  of  cholera,  and  one  of  them  nar- 
rowly escaped  death. 

Morphology  and  Staining. — The  vibrio  or  spirillum  of  cholera  is 
a  small  curved  rod,  varying  from  one  to  two  micra  in  length.  The 
degree  of  curvature  may  vary  from  the  slightly  bent,  comma-like 
form  to  a  more  or  less  distinct  spiral  with  one  or  two  turns.  The 
spirals  do  not  lie  in  the  same  plane,  being  arranged  in  corkscrew 
fashion  in  three  dimensions.  The  spirillum  is  actively  motile  and 
owes  its  motility  to  a  single  polar  flagellum,  best  demonstrated  by 
Van  Ermengem's  flagella  stain.  Spores  are  not  found.  In  young 
cultures  the  comma  shapes  predominate,  in  older  growths  the  longer 
forms  are  more  numerous.  Strains  which  have  been  cultivated 
artificially  for  prolonged  periods  without  passage  through  the  animal 
body  have  a  tendency  to  lose  the  curve,  assuming  a  more  bacillus- 
like  appearance.  The  spirilla  are  stained  with  all  the  usual  aqueous 
anilin  dyes.  They  are  decolorized  by  Gram's  method.  In  histological 

,  Deut.  med.  Woch.,  1883  and  1884. 

831 


832  PATHOGENIC  MICROORGANISMS 

section  they  are  less  easily  stained,  but  may  be  demonstrated  by 
staining  with  alkaline  methylene  blue. 

Cultivation. — The  cholera  spirillum  grows  easily  upon  all  the 
usual  culture  media,  thriving  upon  meat-extract  as  well  as  upon 
meat-infusion  media.  Moderate  alkalinity  of  the  media  is  prefer- 
able, though  slight  acidity  does  not  prevent  growth. 

In  gelatin  plates  growth  appears  at  room  temperature  within 
twenty-four  hours  as  small,  strongly  refracting  yellowish-gray,  pin- 
head  colonies.  As  growth  increases  the  gelatin  is  fluidified.  Under 


& 


\  '  V      '«? 

"  "*/V>**-: 


^-' 


/. 


>^f 

«?> 


-gf^-Wr'- 

^V/Pflt      CB*^!    ^     /    'A 


FIG.  87. — CHOLERA  SPIRILLUM.     (After  Frankel  and  Pfeiffer.) 

magnification  these  colonies  appear  coarsely  granular  with  margins 
irregular  because  of  the  liquefaction.  Liquefaction,  too,  causes  a 
rapid  development  in  such  colonies  of  separate  concentric  zones  of 
varying  refractive  power.  Old  strains,  artificially  cultivated  for 
long  periods,  lose  much  of  their  liquefying  power. 

In  gelatin  stab  cultures  fluidification  begins  at  the  surface,  rapidly 
giving  rise  to  the  familiar  funnel-shaped  excavation. 

Upon  agar  plates,  within  eighteen  to  twenty-four  hours,  grayish, 
opalescent  colonies  appear,  which  are  as  a  rule  easily  differentiated 
by  their  transparency  from  the  other  bacteria  apt  to  appear  in  feces. 
Agar  plates,  therefore,  are  important  in  the  isolation  of  these 
organisms. 


ASIATIC   CHOLERA  AND   THE   CHOLERA  ORGANISM          833 

Coagulated  blood  serum  is  liquefied  by  the  cholera  vibrio.  On 
potato,  growth  is  profuse  and  appears  as  a  brownish  coarse  layer.  In 
milk,  growth  is  rapid  and  without  coagulation.  In  broth,  general 
clouding  and  the  formation  of  a  pellicle  result.  The  rapidity  and 
luxuriance  of  growth  of  the  cholera  spirillum  upon  alkaline  pepton 
solutions  render  such  solutions  peculiarly  useful  as  enriching  media 
in  isolating  this  microorganism  from  the  stools  of  patients.  In 
pepton  solution,  too,  the  cholera  spirillum  gives  rise  to  abundant 
indol,  demonstrated  in  the  so-called  " cholera-red"  reaction.  This 
reaction  has  a  distinct  diagnostic  value,  but  is  by  no  means  specific. 
In  the  case  of  the  cholera  spirillum  the  mere  addition  of  strong 
sulphuric  acid  suffices  to  bring  out  the  color  reaction.  This  is  due 


FIQ.  88.  FIG.  89. 

FIG.  88.— CHOLERA  SPIRILLUM.    Stab  Culture  in  Gelatin,  three  days  old. 
FIG.  89. — CHOLERA  SPIRILLUM.     Stab  Culture  in  Gelatin,  six  days  old.     (After 
Frankel  and  Pfeiffer.) 

to  the  fact  that,  unlike  some  other  indol-producing  bacteria,  the 
cholera  organism  is  able  to  reduce  the  nitrates  present  in  the  medium 
to  nitrites,  thus  itself  furnishing  the  nitrite  necessary  for  the  color 
reaction.  The  medium  which  is  most  suitable  for  this  test  is  that 
proposed  by  Dunham,2  consisting  of  a  solution  of  1  per  cent  of 
pure  pepton  and  .5  per  cent  NaCl  in  water. 

Dieudonne  has  recommended  a  selective  medium  upon  which 
cholera  spirilla  will  grow  well,  but  upon  which  the  colon  bacillus 
will  grow  either  very  sparsely  or  not  at  all.  Cocci  will  produce 
minute  pin-point  colonies  only  and  other  common  bacilli  like  those 
of  the  proteus  group  will  grow  hardly  more  easily  than  bacillus 
coli.  Its  preparation  is  very  simple. 


2  Dunham,  Zeit.  f.  Hyg.,  ii,  1887. 
'Dieudonne  A.,  Cent.  Bakt.,  1.,  orig.,  1909, 


834  PATHOGENIC   MICROORGANISMS 

To  seventy  parts  of  ordinary  3  per  cent  agar,  neutralized  to  litmus,  there 
are  added  thirty  parts  of  a  sterile  mixture  of  equal  parts  of  defibrinated 
beef  blood  and  normal  sodium  hydrate. 

The  latter  is  sterilized  by  steam  before  being  added  to  the  agar.  This 
pure  alkali  agar  is  poured  out  in  plates  and  allowed  to  dry  several  days  at 
37°  or  five  minutes  at  60°. 

The  material  to  be  examined  is  smeared  upon  the  surface  of  these 
plates  with  a  glass  rod.  If  the  blood-alkali  mixture  is  prepared 
beforehand  and  allowed  to  stand  for  four  or  five  weeks,  the  plates 
may  be  used  immediately  after  pouring  (Teague). 

The  principle  of  this  medium  is  that  cholera  will  grow  in  the 
presence  of  an  amount  of  alkali  which  inhibits  other  fecal  bacteria. 

For  other  cholera  media  see  the  section  on  media  in  the  first  part 
of  this  book. 

The  rational  basis  for  the  isolation  of  cholera  spirilla  from  fecal 
or  other  material  is  found  in  two  chief  properties  of  the  spirilla. 
These  have  been  described  to  us  by  Teague  as  follows:  (1)  It  grows 
on  media  of  an  alkalinity  that  retards  or  completely  inhibits  the 
growth  of  most  of  the  fecal  bacteria.  (2)  It  comes  to  the  surface 
of  fluid  media,  rich  in  oxygen,  to  a  greater  extent  than  do  the 
fecal  bacteria. 

The  best  results  in  the  practical  isolation  of  the  cholera  spirilla 
from  stools  are  obtained  by  making  use  of  both  of  these 
properties  from  the  beginning.  A  portion  of  the  stool  is  seeded 
directly  into  alkalin-pepton  water.  The  broth  used  should  be 
distinctly  alkalin,  titrated  to  —0.5,  or  —1.0,  with  phenolphthalein. 
After  six  to  twelve  hours,  a  loopful  from  the  surface  of  these  pepton 
water  tubes  is  plated  upon  plates  of  Dieudonne's  medium,  and  is 
also  transferred  to  a  second  series  of  alkalin-pepton  water  tubes. 
Once  isolated,  the  spirilla  are  identified  by  their  morphology  and 
motility,  by  the  appearance  of  their  colonies,  by  their  manner  of 
growth  upon  gelatin  stabs,  by  the  cholera-red  reaction,  and,  finally, 
by  agglutinative  tests  in  immune  sera.  Owing  to  the  existence  of 
other  spirilla  morphologically  and  culturally  similar,  the  serum  reac- 
tions are  the  only  absolutely  positive  differential  criteria. 

For  isolation  of  the  bacteria  from  water,  it  is,  of  course,  necessary 
to  use  comparatively  large  quantities.  Fliigge  and  Bitter  advise 
the  distribution  of  about  a  liter  of  water  in  ten  or  twelve  Erlen- 
meyer  flasks.  To  each  of  these  they  add  10  c.c.  of  sterile  pepton-salt 
solution  (pepton  ten  per  cent,  NaCl  five  per  cent).  After  eighteen 


ASIATIC  CHOLERA  AND  THE  CHOLERA  ORGANISM          835 

hours  at  37.5°  C.  the  surface  growths  in  these  flasks  are  examined 
both  microscopically  and  culturally  as  before. 

Biological  Considerations. — The  cholera  spirillum  is  aerobic  and 
facultatively  anaerobic.  It  does  not  form  spores.  The  optimum 
temperature  for  its  growth  is  about  37.5°  C.  It  grows  easily,  how- 
ever, at  a  temperature  of  22°  C.  and  does  not  cease  to  grow  at 
temperatures  as  high  as  40°.  Frozen  in  ice,  these  bacteria  may 
live  for  about  three  or  four  days.  Boiling  destroys  them  imme- 
diately. A  temperature  of  60°  C.  kills  them  in  an  hour.  In  impure 
water,  in  moist  linen,  and  in  food  stuffs,  they  may  live  for  many 
days.  Associated  with  saprophytes  in  feces  and  other  putrefying 
material,  and  wherever  active  acid  formation  is  taking  place,  they 
are  destroyed  within  several  days.  Complete  drying  kills  them  in 
a  short  time.  The  common  disinfectants  destroy  them  in  weak 
solutions  and  after  short  exposures  (carbolic  acid,  five-tenths  per 
cent  in  one-half  hour ;  bichlorid  of  mercury,  1 :100,000  in  ten  minutes ; 
mineral  acids,  1:5,000  or  10,000  in  a  few  minutes). 

Cholera  in  Man. — In  man  the  disease  is  contracted  by  ingestion  of 
cholera  organisms  with  water,  food,  or  any  contaminated  material.  The 
disease  is  essentially  an  intestinal  one.  The  bacteria,  very  sensitive  to 
an  acid  reaction,  may  often,  if  in  small  numbers,  be  checked  by  the 
normal  gastric  secretions.  Having  once  passed  into  the  intestine, 
however,  they  proliferate  rapidly,  often  completely  outgrowing  the 
normal  intestinal  flora.  Fatal  cases,  at  autopsy,  show  extreme  con- 
gestion of  the  intestinal  walls.  Occasionally  ecchymosis  and  localized 
necrosis  of  the  mucosa  may  be  present  and  swelling  of  the  solitary 
lymph-follicles  and  Peyer's  patches.  Microscopically  the  cholera 
spirilla  may  be  seen  to  have  penetrated  the  mucosa  and  to  lie  within 
its  deepest  layers  close  to  the  submucosa.  The  most  marked  changes 
usually  take  place  in  the  lower  half  of  the  small  intestine.  The 
intestines  are  filled  with  the  characteristically  fluid,  slightly  bloody, 
or  "rice-water"  stools,  from  which  often  pure  cultures  of  the  cholera 
vibrio  can  be  grown.  The  microorganisms  can  be  cultivated  only 
from  the  intestines  and  their  contents,  and  the  parenchymatous 
degenerations  taking  place  in  other  organs  must  be  interpreted  as 
being  purely  of  toxic  origin. 

Tli ere  is  at  the  same  time  a  profound  toxemia  due,  in  part,  at 
least  to  the  absorbed  cholera  substances. 

The  incubation  time  of  the  disease  is  usually  short,  lasting  from 
a  few  hours  to  several  days.  The  disease  usually  begins,  with  diar- 


836  PATHOGENIC   MICROORGANISMS 

rhea  which  gradually  becomes  more  violent  until  the  colorless 
typical,  rice  water  stools  appear.  Castellani  and  Chalmers  describe 
the  further  course  as  follows: 

"Vomiting  generally  appears  early,  food  being  first  expelled,  fol- 
lowed later  by  watery  fluid  with  which  bile  and  occasionally  blood 
may  be  mixed.  As  the  purging  and  vomiting  persist  the  urine 
diminishes  and  may  stop,  and  fluid  departs  from  the  subcutaneous 
tissues,  which  therefore  contract  so  that  the  face  alters,  the  nose 
becoming  sharp,  the  cheekbones  prominent,  and  eyes  sunken  and  the 
skin  of  the  fingers  becomes  wrinkled  like  that  of  a  washerwoman. ' ' 

A  considerable  role  is  played  in  the  subsequent  course  of  the 
disease  by  the  depletion  of  water,  with  consequent  aneuria,  low  blood 
pressure,  cyanosis,  acidosis,  etc.  The  therapeutic  effect  of  saline 
infusions  is  said  to  be  astonishing. 

Animal  Pathogenicity. — In  animals,  cholera  never  appears  as  a 
spontaneous  disease.  Nikati  and  Rietsch4  have  succeeded  in  produc- 
ing a  fatal  disease  in  guinea-pigs  by  opening  the  peritoneum  and 
injecting  cholera  spirilla  directly  into  the  duodenum.  Koch5  suc- 
ceeded in  producing  a  fatal  cholera-like  disease  in  animals  by  in- 
troducing infected  water  into  the  stomach  through  a  catheter  after 
neutralization  of  the  gastric  juice  with  sodium  carbonate.  At  the 
same  time,  he  administered  opium  to  prevent  active  peristalsis.  A 
method  of  infection  more  closely  analogous  to  the  infection  in  man 
was  followed  by  Metchnikoff,  who  successfully  produced  fatal  dis- 
ease in  young  suckling  rabbits  by  contaminating  the  maternal  teat. 

Subcutaneous  inoculation  of  moderate  quantities  of  cholera 
spirilla  into  rabbits  and  guinea-pigs  rarely  produces  more  than  a 
temporary  illness.  Intraperitoneal  inoculation,  if  in  proper  quan- 
tities, generally  leads  to  death.  It  will  be  remembered  that  when 
working  with  intraperitoneal  cholera  inoculations  the  phenomenon 
of  bacteriolysis  was  discovered  by  Pfeiffer. 

Different  strains  of  cholera  spirilla  vary  greatly  in  their  virulence. 
The  virulence  of  most  of  them,  however,  can  be  enhanced  by  re- 
peated passages  through  animals.  Most  of  our  domestic  animals 
enjoy  considerable  resistance  against  cholera  infection,  though  under 
experimental  conditions  successful  inoculations  upon  dogs,  cats,  and 
mice  have  been  reported.  Doves  are  entirely  insusceptible. 


4  Nikati  und  Eietsch,  Deut.  med.  Woch.,  1884. 

5  Koch,  Deut.  med.  Woch.,  1885. 


ASIATIC  CHOLERA  AND  THE  CHOLERA  ORGANISM          837 

Cholera  Toxin. — The  absence  of  the  cholera  spirilla  from  the 
internal  organs  of  fatal  cases,  in  spite  of  the  severe  general  symp- 
toms of  the  disease,  points  distinctly  to  the  existence  of  a  strong 
poison  produced  in  the  intestine  by  the  microorganisms  and  absorbed 
by  the  patient.  It  was  in  this  sense,  indeed,  that  Koch  first  inter- 
preted the  clinical  picture  of  cholera.  Numerous  investigations  into 
the  nature  of  these  toxins  have  been  made,  the  earlier  ones  defective 
in  that  definite  identification  of  the  cultures  used  for  experimentation 
were  not  carried  out. 

Pfeiffer,6  in  1892,  was  able  to  show  that  filtrates  of  young  bouillon 
cultures  of  cholera  spirilla  were  but  slightly  toxic,  whereas  the  dead 
bodies  of  carefully  killed  agar  cultures  were  fatal  to  guinea-pigs 
even  in  small  quantities.  In  consequence,  he  regarded  the 
cholera  poison  as  consisting  chiefly  of  an  endotoxin.7  The  opinion 
as  to  the  endotoxic  nature  of  the  cholera  poison  is  not,  however, 
shared  by  all  workers.  Metchnikoff,  Roux,  and  Salimbeni,8  in  1896, 
succeeded  in  producing  death  in  guinea-pigs  by  introduction  into 
their  peritoneal  cavities  of  cholera  cultures  enclosed  in  celloidin 
sacs.  Brau  and  Denier,9  and,  more  recently,  Kraus,10  claim  that 
they  have  succeeded  not  only  in  demonstrating  a  soluble  toxin  in 
alkaline  broth  cultures  of  cholera  spirilla,  but  in  producing  true 
antitoxins  by  immunization  with  such  cultures.  It  appears,  there- 
fore, that  the  poisonous  action  of  the  cholera  organisms  may  depend 
both  upon  the  formation  of  true  secretory  toxins  and  upon  endo- 
toxins.  Which  of  these  is  paramount  in  the  production  of  the 
disease  can  not  be  at  present  definitely  stated.  In  favor  of  the 
great  importance  of  the  endotoxic  elements  is  the  failure,  thus  far, 
to  obtain  successful  therapeutic  results  with  supposedly  antitoxic 
sera. 

Epidemiology. — Cholera  is  essentially  a  disease  of  man.  Endemic 
in  India  and  other  Eastern  countries,  it  has  from  time  to  time 
epidemically  invaded  large  territories  of  Europe  and  Asia,  not  in- 
frequently assuming  pandemic  proportions  and  sweeping  over  almost 
the  entire  earth.  Five  separate  cholera  epidemics  of  appalling  mag- 
nitude occurred  during  the  nineteenth  century  alone;  several  of 

6  Pfeiffer,  Zeit.  f.  Hyg.,  xi,  1892. 

''Pfeiffer  und  Wassermann,  Zeit.  f.  Hyg.,  xiv,  1893. 

8  Metchnikoff,  Eoux,  et  Salimbeni,  Ann!  de  Pinst.  Pasteur,  1896. 

9  Brau  et  Denier,  Comptes  rend,  de  Pacad.  des  sci.,  1906. 

10  E.  Kraus,  Cent.  f.  Bakt.,  1906. 


838  PATHOGENIC   MICROORGANISMS 

these,  spreading  from  India  to  Asia  Minor,  Egypt,  Russia,  and  the 
countries  of  Central  Europe,  reached  even  to  North  and  South 
America.  The  last  great  epidemic  began  about  1883,  traveled  grad- 
ually westward,  and  in  1892  reached  Germany  where  it  appeared 
with  especial  virulence  in  Hamburg,  and  thence,  following  the  high- 
ways of  ocean  commerce,  entered  America  and  Africa.  During  this 
epidemic  in  Russia  alone  800,000  people  fell  victims  to  the  disease. 

An  important  epidemiological  fact  is  the  existence  of  certain 
endemic  foci  where  cholera  is  always  going  on  and  from  which 
epidemics  and  pandemics  originate.  The  chief  endemic  focus 
seems  to  be  located  in  lower  Burmah,  and  it  is,  as  yet,  an 
unsolved  puzzle  as  to  why  the  disease  should  remain  smouldering 
in  such  regions  and  spread  widely  only  at  certain  periods,  five,  ten 
or  more  years  apart.  During  recent  years  important  epidemics 
have  occurred  between  the  years  1879  and  1910.  In  1879  an 
epidemic  spread  to  Europe  through  Egypt  and  this  outbreak  is 
notable  because  in  1883  in  Egypt,  Koch,11  as  head  of  the  German 
Cholera  Commission,  isolated  the  cholera  spirillum.  In  1891 
another  great  epidemic,  originating  in  India,  is  stated  by  Cas- 
tellani  as  having  started  on  the  occasion  of  a  bathing  festival 
held  on  the  Ganges.  It  spread  among  pilgrims  and  reached  Europe 
in  1892,  appearing  with  particular  virulence  in  Hamburg.  From 
there  it  spread  to  America  and  Africa  by  ocean  commerce. 
During  this  epidemic  it  is  said  that  800,000  people  fell  victims 
in  Russia  alone.  Violle  records  that  in  1908,  1909  and  1910 
there  were  a  series  of  epidemics  in  Russia.  In  1908  there  were 
about  30,000  cases  with  14,000  deaths;  in  1909,  there  were  21,000 
cases  with  9,700  deaths,  and  in  1909  to  1910  there  were  130,000 
deaths.  During  the  Balkan  War  in  1912  cholera  appeared 
among  the  armies.  During  the  late  war  there  were  cases  of  cholera 
in  Galicia  in  the  Austrian  Army,  and  there  were  outbreaks  in  Bul- 
garia, Greece  and  Turkey,  and  in  Mesopotamia. 

The  prevalence  of  cholera  as  an  important  epidemic  disease  may 
be  estimated  by  the  following  chart  of  cholera  epidemics  of  the 
last  hundred  years  which  is  taken  from  Violle 's  recent  work  on 
cholera  (1918)  to  which  numerous  references  have  been  made. 

The  disease  always  originates  from  the  dejecta  of  cholera  patients 
and  carriers.  At  times  of  epidemic,  infection  of  the  water  and 

11  Koch,  Deut.  med.  Woch.,  1883  and  1884. 


ASIATIC   CHOLERA  AND  THE  CHOLERA  ORGANISM 


839 


food  supplies  naturally  plays  an  important  role,  and  in  such  epi- 
demics, as  the  one  in  Hamburg,  the  water  supply  was  primarily 
responsible.  The  distribution  of  the  disease  here  followed  definitely 

EPIDEMICS  OF  CHOLERA  IN  VARIOUS  PLACES  l 


Place 

Date 

Deaths 

Havana  

1833 

8,000 

Malta.. 

1837 

4,000 

London  

1832 

4,000 

Paris.    ...                    .... 

1832 

7,000 

Basra 

1821 

5000 

Lahore  

1845 

22,000 

Tabriz 

1852 

12  000 

Teheran  
Bagdad 

1852 
1852 

15,000 
2000 

Bellary  and  Mysore,  India  
Province  of  Bombay 

1865 
1865 

40,000 
84,000 

Cachemire  

1892 

5,000 

England  

1854 

20,000 

France 

1854 

140  000 

Italy  

1854 

24,000 

Egvpt  . 

1865 

60000 

Eevpt 

1883 

50000 

Egypt.  .  , 

1831 

150  000 

Cairo 

1831 

36000 

Cairo  

1902 

33000 

Russia 

1909-10 

130  000 

Rosetta  

1865 

2,168 

Epidemic  of  Cholera  in  Russia 
In  1908,  30,000  cases 

14000 

In  1909,  21,000  cases  

9,700 

the  distribution  of  the  infected  water  supply  and  the  organisms 
were  isolated  from  the  water.  This  epidemic  is  one  of  the  classical 
water  epidemics  and  has  served  more  than  any  other  water  epidemic 
in  impressing  medical  and  health  authorities  with  the  importance 
of  water  supply  supervision.  In  countries  like  India  and  Egypt, 
etc.,  where  water  supplies  are  often  taken  from  collecting  tanks  not 
properly  supervised,  and  from  individual  wells,  and  where  the  super- 
vision of  feces  disposal  is  not  strict,  it  is  quite  natural  that  dis- 
tribution by  water  supplies  should  be  extremely  important.  Violle 
adds  a  number  of  interesting  instances  of  water  transmission  which 
occurred  in  France  in  1885,  in  which  the  source  was  soiled  linen 


840  PATHOGENIC   MICROORGANISMS 

washed  in  a  stream  with  distribution  of  the  disease  further  down- 
stream in  other  villages,  but  not  in  any  of  the  villages  higher  up 
the  river.  It  is  also  probable  that  in  countries  such  as  India,  the 
custom  of  throwing  dead  bodies  into  rivers  may  contribute  ma- 
terially to  the  constant  presence  of  the  disease. 

In  endemic  centers,  it  is  more  than  likely  that  the  cholera  carrier 
is  a  very  important  factor  of  distribution.  The  existence  of  the 
carrier  is  proven  beyond  doubt,  and,  as  in  typhoid,  individuals  may 
remain  carriers  for  very  long  periods.  Greig  has  shown  that  the 
organisms  may  live  in  the  gall-bladders  of  human  beings  as  in  the 
typhoid  carrier  state.  McLaughlin12  has  found  as  many  as  7  per 
cent  of  the  population  of  an  infected  district  to  be  cholera  carriers. 

Again,  as  in  typhoid,  distribution  of  fecal  material  to  food  by 
flies  probably  plays  a  very  important  role,  and  according  to  Barber 
the  organisms  may  live  for  some  time  in  the  intestines  in  such 
insects  as  cockroaches.  Whether  or  not  domestic  animals  can  act 
as  distributers  of  the  organisms  is  uncertain.  Violle  quotes  Haffkine 
as  stating  that  he  had  found  the  spirilla  in  the  intestines  of  cattle, 
and  that  they  were  found  by  Hahn  in  the  intestines  of  cows  during 
cholera  epidemics.  The  importance  of  this,  however,  is  still  quite 
uncertain. 

In  nature  the  cholera  spirilla  may,  under  favorable  conditions, 
remain  alive  for  considerable  periods.  In  drinking  water  they  have 
been  found  alive  after  several  days  and  they  may  remain  alive  for 
weeks  in  water  supplies.  From  the  investigations  of  Wernieke,18 
Shirnoff14  and  others  it  would  appear  that  under  favorable  condi- 
tions the  spirilla  may  remain  alive  in  river  water  and  other  natural 
waters  for  weeks  or  even  months.  In  milk  and  other  foods,  the 
longevity  of  the  cholera  spirilla  seems  to  depend  particularly  upon 
the  nature  and  numbers  of  other  bacteria  present  and  on  the  produc- 
tion of  an  acid  reaction.  In  cholera  stools  they  will  remain  alive 
until  considerable  putrefaction  has  taken  place  and,  therefore,  may 
be  assumed  under  favorable  conditions  to  live  at  least  one  day, 
or  perhaps  three  days  or  longer.  In  cold  weather  when  bacterial 
growth  is  more  or  less  inhibited,  they  may  remain  alive  much  longer 
than  this. 

12  McLaughlin,  quoted  from  Eosenau  's  Preventive  Medicine  and  Hygiene,  D. 
Appleton  and  Co.,  New  York  and  London,  1921. 

13  Werniclce,  Hyg.  Eundschau,  1895. 
uShirnoff,  Cent.  f.  Bakt.,  41,  1908. 


ASIATIC   CHOLERA   AND   THE   CHOLERA  ORGANISM          841 

Cholera  Immunization. — One  attack  of  cholera  confers  protection 
against  subsequent  infection.  Active  immunization  of  animals  may 
be  accomplished  by  inoculation  of  dead  cultures,  or  of  small  doses 
of  living  bacteria.  In  the  serum  of  immunized  animals  specific 
bacteriolytic  and  agglutinating  substances  are  found.  The  discovery 
of  bacteriolytic  immune  bodies,  in  fact,  was  made  by  means  of 
cholera  spirilla.  Both  the  bacteriolysins  and  the  agglutinins,  be- 
cause of  their  specificity,  are  of  great  importance  in  making  a  bac- 
teriological diagnosis  of  true  cholera  organisms. 

Prophylactic  Vaccination. — Active  immunization  of  cholera  was 
one  of  the  first  methods  of  prophylactic  vaccination  attempted  in 
the  bacteriological  era  of  infectious  disease  study.  The  work  was 
done  by  a  Spanish  bacteriologist,  Ferran,  who  had  been  a  pupil  of 
Pasteur,  and  as  early  as  1884  carried  out  immunization  experiments 
with  cholera  on  guinea.-pigs.  Ferran,15  in  accordance  with  the 
methods  prevalent  at  that  time,  worked  with  attenuated  cholera 
cultures  and  developed  a  method  of  attenuation  which  depended 
upon  room-temperature  cultivation  on  gelatin.  He  tried  this  method 
on  human  beings  in  Spain  in  1885  with  results  which  seemed  to  him 
encouraging.  Subsequent  to  this  many  different  vaccines  have  been 
developed.  Haffkine16  worked  intensely  on  the  subject  and  observed 
the  results  of  vaccination  on  an  enormous  number  of  people  in 
India,  over  a  period  of  more  than  ten  years.  Haffkine 's  virus  has 
undergone  a  number  of  modifications  since  he  first  used  it.  He, 
too,  made  use  of  living  cultures,  beginning  his  experiments  with 
attenuation  of  cholera  spirilla  by  cultivation  at  temperatures  of 
40°  and  over,  using,  at  first,  a  less  virulent  and  next  a  more  virulent 
strain.  Later,  it  was  found  that  the  cultures  attenuated  by  cultiva- 
tion at  increased  temperatures  were  not  necessary,  and  it  appears 
at  the  present  time  that  in  most  places  only  cultures  of  a  virulence 
enhanced  by  passage  through  guinea-pigs  are  used.  The  extensive 
experimental  work  in  India  mentioned  above  seems  to  have  shown 
that  there  is  a  distinct  prophylactic  value  in  the  use  of  Haffkine 's 
virus. 

Other  observers  have  made  use  chiefly  of  killed  cultures.  The 
French  vaccine  made  at  the  Pasteur  Institute  consists  of  broth 
cultures  killed  at  50°.  Kolle17  grows  his  cholera  spirilla  on  agar, 

15  Ferran,  Comptes  rend,  de  1'acad.  des  sciences,  1885. 

"Haffkine,  Bull,  med.,  1892. 

"  Kolle  and  Schurmann,  Kolle  and  Wassermann  Handb.,  Vol.  4,  Second  Edition. 


842  PATHOGENIC   MICROORGANISMS 

suspending  them  in  salt  solution,  killing  at  56°  C.  for  one  hour, 
then  adding  one-half  per  cent  carbolic  acid.  Other  observers,  like 
Nicoll  and  Vincent18  killed  without  heat,  by  the  addition  of  carbolic 
acid.  Extracts  of  the  cholera  spirilla  have  also  been  used  in  various 
ways.  Strong19  grows  cholera  organisms  on  agar,  takes  them  up 
in  salt  solution,  kills  at  60°  and  then  allows  the  suspensions  to  stand 
in  the  incubator  for  about  five  days,  subsequently  filtering  through 
a  Berkefeld  candle.  This  filtrate  is  used  for -inoculation,  after  its 
sterility  has  been  determined  by  culture.  Wassermann20  has  used 
materials  prepared  by  precipitation  of  cultures  with  alcohol.  Cas- 
tellani21  during  the  last  ten  years  has  prepared  what  he  calls  a 
T.  A.  B.  C.,  or  tetravaccine,  which  is  made  by  mixing  agar  cultures 
of  typhoid,  paratyphoid  "A,"  paratyphoid  "B,"  and  cholera  in 
saline  emulsion.  The  emulsion  is  killed  with  one-half  per  cent 
carbolic  acid,  preserved  for  twenty-four  hours  in  this  form  at  room 
temperature  and  then  standardized  by  the  usual  counting  chamber 
method  so  that  1  c.c.  should  contain  five  hundred  thousand  typhoid, 
250  thousand  paratyphoid  "A,"  250  thousand  paratyphoid  "B," 
and  two  thousand  million  cholera  spirilla.  0.5  c.c.  of  this  is  injected, 
three  doses  being  given  within  two  weeks.  This  is  the  vaccine  which 
we  used  on  the  Serbian  Army  during  the  war. 

The  principle  underlying  all  these  procedures  seems  to  us  to 
be  the  same,  in  that  they  consist  of  introducing,  subcutaneously, 
substances  derived  from  the  bodies  of  cholera  spirilla.  And  since 
the  cholera  organisms  probably  do  not  live  very  long  after  sub- 
cutaneous introduction,  it  is  not  likely  that  it  makes  very  much 
difference  whether  attenuated  living  cultures,  or  dead  cultures  are 
used. 

As  far  as  the  available  statistics  show  at  the  present  time,  cholera 
vaccination  is  of  distinct  value.  This  has  been  the  judgment  of 
those  who  have  scrutinized  Haffkine's  immunization  experiments, 
as  well  as  those  who  have  observed  more  recent  army  experiences. 
The  following  table  which  we  used  in  our  Nelson  article,  again 
taken  from  Violle,  will  give  some  idea  of  the  comparisons  made 


18  Nicoll  and  Vincent.     Cited  from  Violle  loc.  cit. 

19  Strong.     J.  of  Exp.  Med.,  Vol.  8,  p.  229,  1905. 

20  Wassermann.     Festschr.     E.  Koch,  Jena,  1903. 

21  Castellani  and   Chalmers,   Manual   of   Tropical   Medicine,   W.    Wood  &  Ho, 
New  York,   1919. 


ASIATIC   CHOLERA  AND  THE  CHOLERA  ORGANISM 


843 


upon  vaccinated  and  unvaccinated  individuals'  among  troops,  and 
in  some  of  the  more  recent  experiments. 

I 

EPIDEMIC    OF    CHOLERA    IN    THE    GREEK    ARMY    DURING    THE 
SECOND  BALKAN  WAR  (from  ARNAUD) 


Number  of 
Soldiers 


Cases  of  Cholera, 
Per  Cent 


Vaccinated,  2  inoculations 76,652  0.43 

Vaccinated,  1  inoculation 21,216  3 . 12 

Not  vaccinated 14,332  5.7 

Morbidity,  1,801  (12.5  per  cent) 
Mortality,  348  (2.5  per  cent) 

II 

EPIDEMIC  OF  CHOLERA  IN  RUSSIA,   1912   (from  ABRAMOW) 

Numbers  of  Cases  of  Cholera, 

Soldiers  Per  Cent 

Vaccinated 1,500  5  =  0.3 

Not  vaccinated 

Ill 
RECENT    EPIDEMIC    OF    CHOLERA    IN    INDIA    (from  (KATRINE) 

Cases  of  Cholera      Deaths,  Per  Cent 

Not  inoculated "I  11 

Inoculated /        8'°°  3 

IV 

EPIDEMIC    OF    CHOLERA    IN    THE    GREEK    ARMY  DURING    THE 
THE  BALKAN  WAR   (from  SAVAS) 

Number  of  Deaths, 

Individuals  Per  Cent 

Not  vaccinated }  20 

Vaccinated  once \      10,000  3 

Vaccinated  twice J  1 


Cholera  vaccination  naturally  is  of  relative  value  only,  just  as 
this  is  the  case  in  typhoid  vaccination.  Vaccination  must  be  repeated 
certainly  every  two  years  and  probably  more  often  in  the  case  of 
armies  in  the  field. 


844  PATHOGENIC   MICROORGANISMS 

CHOLERA-LIKE   SPIRILLA 

The  biological  group  of  the  spirilla,  to  which  the  cholera 
spirillum  belongs,  is  a  large  one,  numbering  probably  over  a  hundred 
separate  species.  Most  of  these  are  of  bacteriological  importance 
chiefly  because  of  the  difficulties  which  they  add  to  the  task  of 
differentiation,  for  while  some  of  them  simply  bear  a  morphological 
resemblance  to  the  true  cholera  vibrio,  others  can  be  distinguished 
only  by  their  serum  reactions  and  pathogenicity  for  various  animals. 
Additional  difficulty,  too,  is  contributed  by  the  fact  that  within  the 
group  of  true  cholera  organisms  occasional  variations  in  agglu- 
tinability  and  bacteriolytic  reactions  may  exist.  Certain  strains, 
too,  the  six  El  Tor  cultures  isolated  by  Gottschlich,  while  in  every 
respect  similar  to  true  cholera  spirilla,  are  considered  as  a  separate 
sub-species  by  Kraus,22  because  of  their  ability  to  produce  hemolytic 
substances,  a  function  lacking  in  other  cholera  strains. 

Spirillum  Metchnikovl — This  spirillum  was  discovered  by 
Gamaleia23  in  the  feces  and  blood  of  domestic  fowl,  in  which  it  had 
caused  an  intestinal  disease.  Morphologically  and  in  staining  reac- 
tions it  is  identical  with  Spirillum  choleraeasiaticse.  It  possesses  a 
single  polar  flagellum,  and  is  actively  motile.  Culturally  it  is  iden- 
tical with  Vibrio  cholerae  except  for  slightly  more  luxuriant  growth 
and  more  rapid  fluidification  of  gelatin.  It  gives  the  cholera-red 
reaction  in  pepton  media. 

It  is  differentiated  from  the  cholera  vibrio  by  its  power  to 
produce  a  rapidly  fatal  septicemia  in  pigeons  after  subcutaneous 
inoculation  of  minute  quantities.24  It  is  much  more  pathogenic  for 
guinea-pigs  than  the  cholera  vibrio.  It  is  not  subject  to  lysis  or 
agglutinated  by  cholera  immune  sera. 

Spirillum  Massaua. — This  organism  was  isolated  at  Massaua  by 
Pasquale25  in  1891  from  the  feces  of  a  clinically  doubtful  case  of 
cholera.  Culturally  and  morphologically  it  is  much  like  the  true 
cholera  vibrio,  but  in  pathogenicity  is  closer  to  Spirillum  Metchni- 
kovi,  in  that  small  quantities  produce  septicemia  in  birds.  It  pos- 
sesses four  flagella.  It  does  not  give  a  specific  serum  reaction  with 
cholera  immune  serum. 

22  Kraus,  Kraus  and  Levaditi,  "Handbueh,"  vol.  i.  p.  186. 

23  Gamaleia,  Ann.  de  1'inst.  Pasteur,  1883. 
2*Pfeiffer  und  Nocht,  Zeit.  f.  Hyg.,  vii,  1889. 

25  Pasquale,  Giorn,  med.  de  r.  escre.  ed.  B.  Marina,  Boma,  1891. 


ASIATIC   CHOLERA  AND  THE   CHOLERA  ORGANISM         845 

Spirillum  of  Finkler-Prior.26 — Isolated  by  Finkler  and  Prior  from 
the  feces  of  a  case  of  cholera  nostras.  Morphologically  it  is  like 
the  true  cholera  spirillum,  though  slightly  larger  and  less  uniformly 
curved.  Culturally  it  is  much  like  the  cholera  vibrio,  but  grows 
more  rapidly  and  thickly  upon  the  usual  media.  It  does  not  give 
the  cholera-red  reaction,  nor  does  it  give  specific  serum  reactions 
with  cholera  immune  serum. 

Spirillum  Deneke.27 — A  vibrio  isolated  by  Deneke  from  butter. 
Much  like  that  of  Finkler-Prior.  It  does  not  give  the  cholera-red 
reaction. 

™FinTder  und  Prior,  Erganz.  Hefte,  Cent.  f.  allg.  ges.  Phys.,  1884. 
27  Deneke,  Deut.  med.  Woch.,  iii,  1885. 


CHAPTER   XLIII 

DISEASES  CAUSED  BY  SPIEOCH^ETES  (TEEPONEMATA),  CLASSI- 
FICATION, SYPHILIS  AND  TEEPONEMA  PALLIDUM,  EELAPS1NG 
FEVEES,  VINCENT'S  ANGINA,  YAWS,  AND  THE  SPIROCHAETE 
PEETENUE,  SPIEOCHvETE  GALLINAEUM,  EAT  BITE  FEVEE,  NON- 
PATHOGENIC  SPIEOCHJETES  OF  THE  HUMAN  BODY. 

THE  microorganisms  known  as  spirochsetes  are  slender,  undu- 
lating, corkscrew-like  threads  which  show  definite  variations  both 
structurally  and  culturally  from  the  bacteria  as  a  class.  Most  im- 
portant among  them  are  the  spirochaete  of  relasping  fever,  Spirochaete 
pallida  of  syphilis,  the  spirillum  of  Vincent,  Spirochaete  refringens, 
Spirillum  gallinarum,  a  microorganism  which  causes  disease  in 
chickens,  Spirochaete  anserina,  which  causes  a  similar  condition  in 
geese,  and  several  species  which  have  been  found  as  parasites,  both 
in  animals  and  in  man,  without  having  definite  etiological  connection 
with  disease. 

CLASSIFICATION    OF    SPIRAL   ORGANISMS 

Classification  of  the  spiral  organisms  in  general  is  still  unsatis- 
factory because  the  difficulties  of  staining  and  cultivation  have  made 
it  impossible  to  apply  to  these  organisms  the  same  exact  criteria 
which  can  be  applied  to  most  species  of  bacteria.  We  may  say,  in 
general,  that  the  word  spirillum  should  be  retained  for  true  bacteria 
of  spiral  form  in  which  the  cell  body  is  rigid  and  motility  is  brought 
about  entirely  by  flagella.  Such,  for  instance,  are  the  spirillum  of 
Asiatic  cholera,  the  spirillum  Metchnokovi,  the  spirillum  Deneke 
and  others. 

The  true  spirochaete  are  probably  not  true  bacteria,  and  we  have 
no  exact  criteria  upon  which  we  can  base  their  classification  with 
the  protozoa.  However,  the  striking  parasitism  of  most  of  them, 
and  certain  features  of  their  immunological  relations  would  suggest 
that  they  either  belong  to,  or  are  very  close  to  protozoa.  Schaudinn, 
the  discoverer  of  the  syphilis  organism,  classified  the  treponema 
pallidum  with  the  protozoa  on  the  basis  of  morphological  study. 
He  believed  that  stained  preparations  often  showed  an  undulating 

846 


DISEASES  CAUSED   BY  SPIROCH^TES  847 

membrane  extending  along  the  long  axis  of  the  microorganisms 
similar  to  that  observed  in  trypanosomes.  He  also  asserted  that 
most  of  the  spiral  forms  reproduce  by  cleavage  along  the  longi- 
tudinal axis.  On  the  other  hand,  Laveran,1  Novy  and  Knapp2  and 
others  maintained  a  close  relationship  of  these  microorganisms  to 
the  true  bacteria.3 

A  review  of  observed  facts  seems  to  show  that  most  of  these 
spiral  organisms  have  the  power  of  multiplication  by  transverse 
fission.  Many  of  them  possess  flagella  and  in  some  of  them  definite 
immune  bodies  can  be  demonstrated  in  the  serum  of  infected  sub- 
jects, similar  to  those  produced  by  bacteria  during  infection.  In 
others  again,  like  the  treponema  pallidum,  no  true  circulating  anti- 
bodies against  the  virulent  parasitic  forms  can  be  found.  Indeed, 
in  syphilis  it  seems  that  immunity  exists  only  so  long  as  the  living 
organisms  still  persist  in  the  body,  an  observation  which  is  entirely 
analogous  to  that  made  with  certain  trypanosomes,  and  with  malaria. 
Also,  with  some  of  them,  transmission  by  an  intermediate  insect 
host  in  which  the  spirilla  undergo  multiplication  has  been  definitely 
shown,  a  state  of  affairs  which  corresponds  with  conditions  in  many 
protozoan  infections.  Kolle  and  Hetsch  favor  a  classification  mid- 
way between  the  protozoa  and  the  bacteria,  a  view  which  is  probably 
as  correct  as  any  that  we  have  any  justification  for  holding  at  the 
present  time. 

Noguchi,4  who  has  had  extensive  experimental  experience  with 
the  spirochaete  has  suggested  the  tentative  classification  which 
follows : 

He  calls  attention  to  the  fact  that  the  term  "spirochaeta"  was 
applied  first  by  Ehrenberg  in  1838  to  a  free  living,  fresh  water 
or  marine  form  of  spiral  organism  which  creeps  along  the  surface 
of  an  object  but  does  not  swim,  divides  by  transverse  fission,  and 
probably  has  nothing  to  do  with  the  organism  to  which  we  now 
apply  this  word. 

Noguchi  divides  the  spiral  organisms  of  the  group  which  we  are 
now  considering  into : 

I.  Cristispira  or  Saprospira. — This  is  a  limited  group  of  motile 
spiral  organisms  which  infest  the  great  crystalline  styles  of  certain 

1  Laveran,  Comptes  rend,  de  1'acad.  des  sei.,  1902  and  1903. 

Novy  and  Knapp,  Jour.  Infec.  Dis.,  3,  190(1. 
'  Kolle  and  Hetsch,  "Die  experimentelle  Bakt.,"  Berlin,  1906. 
*  Noguchi,  Jour.  Exper.  Med.,  27,  1918,  575. 


848  PATHOGENIC  MICROORGANISMS 

mollusca.  The  term  was  first  proposed  by  Gross5  iA  1910.  A  type 
of  these  organisms  is  found  in  oysters  (Spirochaeta  balbianii  (Certes,6) 
1882).  Another  genus  of  the  same  order,  Saprospira,  was  found 
by  Gross  to  exist  in  mussels. 

None  of  these  are  pathogenic  for  higher  animals.  They  are  char- 
acterized by  the  presence  of  a  membraneous  structure  which  resembles 
a  crista  or  ridge  which  runs  spirally  along  the  entire  length  of 
the  body.  The  body  is  chambered,  that  is,  transverse  bands  seem 
to  show  along  its  entire  length.  There  are  no  terminal  filaments 
and  there  seems  to  be  a  strong  flexible  membrane.  Reproduction, 
according  to  Gross,  takes  place  by  multiple  transverse  fission  or 
sporulation,  but  Noguchi  has  failed  to  confirm  the  occurrence  of 
sporulation. 

II.  Spironema  and  Treponema.— This  is  a  large  group  of  parasitic 
spiral  organisms  which  are  commonly  spoken  of  as  the  "spirochete" 
in  medical  nomenclature.  The  characteristic  feature  of  these  is  a 
spiral  flexible  body  with  terminal  filaments,  but  no  undulating  mem- 
brane. They  may  apparently  multiply  by  transverse  as  well  as  by 
longitudinal  fission.  They  move  by  an  undulating  movement,  a  few 
of  them,  however,  retaining  their  regular  curves  during  motion. 
Dobell  in  an  address  before  the  Royal  Society  in  1912  expressed  the 
belief  that  the  word  treponema  should  be  used  for  all  of  the  small 
parasitic  varieties.  Noguchi  believes  with  Gonder  that  the  term 
treponema  should  be  restricted,  as  was  done  by  Schaudinn,7  to  those 
varieties  having  great  constancy  of  curves,  while  spironema  should  be 
applied  to  those  with  less  constant  curves,  but  he,  nevertheless,  classi- 
fies them  together  under  the  same  main  heading  since  he  believes 
they  are  closely  related. 

In  this  class  belong  the  Treponema  pallidum  of  syphilis  and  the 
Treponema  or  Spirochcete  pertenue  of  Yaus.  The  class  also  includes 
the  organisms  of  relapsing  fever,  a  number  of  parasites  found  in 
rodents,  such  as  the  well  known  organism  which  invades  apparently 
normal  mice  (and  was  once  falsely  looked  upon  as  the  cause  of  cancer 
in  mice)  and  various  saprophytic  types  found  in  the  mouth,  intestine 
and  genital  mucous  membranes,  such  as  the  Treponema  calligyrum 


6  Gross,  Mitt.  zool.  Station  Neapel.,   1910-13,  20,  41  and  188,  Cent.  f.  Bakt., 
Orig.,  65,  1912,  83. 

•  Certes,  Bull.  Soc.  zool.  franc.,  7,  1882,  347. 

7  Schaudinn,  Deut.  med.  Woch.,  43,  1909,  1728,  Arb.  a.  d.  k.  Gesundhst.,  1904. 


DISEASES  CAUSED  BY  SPIROCH^TES  849 

found  in  smegma,  the  Treponema  microdentium  and  macrodentium, 
found  in  the  mouth,  especially  under  the  gums,  and  in  the  throat. 

Among  the  Spironema  in  this  main  group  Noguchi  places  the 
Spironema  refringcns8  of  smegma,  the  Spironema  vincenti  of  Vincent's 
angina,  the  Spironema  recurrent^  of  Obermeier,9  the  Spironema 
Duttoni,10  the  Spironema  Kochi,  the  Spironema  gallinarum  and  the 
Spironema  Novyi. 

III.  Leptospira. — The  types  of  this  class  are  the  Leptospira  ictero 
hcemorrJiagice  of  Inada  and  Ido  and  the  Leptospira  icteroidis  recently 
isolated  by  Noguchi  from  cases  of  yellow  fever,  and  probably  repre- 
senting the  etiological  factor  of  that  disease.  These  organisms  are 
much  more  easily  cultivated  than  the  preceding.  They  are  char- 
acterized by  closely  set  regular  spirals  which  remain  unchanged 
during  a  peculiar  rotary  spinning  motion.  As  described  by  Noguchi 
these  organisms,  while  in  motion,  draw  the  entire  body  together 
into  a  straight  line,  except  fcfr  a  hook  formation  of  one  or  both  ends. 
When  one  end  is  extended  and  straight  and  the  other  semicircularly 
hooked,  the  organism  progresses  in  the  sUree'tibn  of  the  straight 
portion,  appearing  to  be  propelled  from  the  rear  by  the  rotary  hook. 
A  specimen  with  both  ends  hooked  remains  stationary  in  spite  of 
its  rapid  rotary  motions.  This  description  is  taken  verbatim  from 
Noguchi.  In  this  sort  of  movement  the  body  assumes  wide  wavy 
undulations.  So  far  no  terminal  or  peritrichal  flagella  have  been 
seen. 

SYPHILIS    AND    SPIROCHJETA   PALLIDA 

(Treponema  pallidum) 

The  peculiar  manifestations  of  syphilis,  its  mode  of  transmission, 
and  the  fact  that  its  primary  lesion  was  always  located  at  the  point 
of  contact  with  a  preceding  case,  have  always  stamped  it  as  unques- 
tionably infectious  in  nature.  Until  very  recently  the  microorgan- 
ism which  gives  rise  to  syphilis  was  unknown.  Many  bacteriologists 
had  studied  the  problem  and  many  microorganisms  for  which 
definite  etiological  importance  was  claimed  had  been  described. 
Most  of  these  announcements,  however,  aroused  little  more  than  a 
sensational  interest  and  received  no  satisfactory  confirmation.  A 

8  Schaudinn  and  Hoffmann,  Arb.  a.  d.  w.  Gesundhst.,  22,  1905. 
8  Obermeier,  Cent,  f .  d.  med.  Wiss.,  11,  1873. 
10  Button  and  Todd,  Brit.  Med.  Jour.,  1905. 


850  PATHOGENIC   MICROORGANISMS 

bacillus  described  by  Lustgarten11  in  1884  seemed,  for  a  time,  to 
have  solved  the  mystery.  The  Lustgarten  bacillus  was  an  acid-fast 
organism  very  similar  to  Bacillus  tuberculosis,  and  found  by  its 
discoverer  in  a  large  number  of  syphilitic  lesions.  The  observation, 
at  first,  aroused  much  interest  and  received  some  confirmation. 
Later  extensive  investigations,  however,  failed  to  uphold  the 
etiological  relationship  of  this  bacillus  to  the  disease  but  identified  it 
with  the  smegma  bacillus,  so  often  a  saprophyte  upon  the  mucous 
membranes  of  the  normal  genitals. 

In  1905,  Schaudinn,12  a  German  zoologist,  working  in  collabora- 
tion with  Hoffmann,  investigated  a  number  of  primary  syphilitic 


FIG.  90. — SPIROCH^JTA  PALLIDA.     Smear  preparation  from  chancre  stained  by  the 

india-ink  method. 

indurations  and  secondarily  enlarged  lymph  nodes,  and  in  both 
lesions  discovered  a  spirochsete  similar  to,  but  easily  distinguished 
from,  the  spirochaetes  already  known.  He  failed  to  find  similar 
microorganisms  in  uninfected  human  beings. 

The  microorganism  described  by  him  as  "Spirochasta  pallida" 
is  an  extremely  delicate  undulating  filament  measuring  from  four 
to  ten  micra  in  length,  with  an  average  of  seven  micra,  and  varying 
in  thickness  from  an  immeasurable  delicacy  to  about  0.5  of  a  micron. 
It  is  thus  distinctly  smaller  and  more  delicate  than  the  spirochaete 
of  relapsing  fever.  Examined  in  fresh  preparations  it  is  actively 
motile,  its  movements  consisting  in  a  rotation  about  the  long  axis, 
gliding  movements  backward  and  forward,  and,  occasionally,  a  bend- 
ing of  the  whole  body.  Its  convolutions,  as  counted  by  Schaudinn, 
vary  from  three  to  twelve  and  differ  from  those  observed  in  many 
other  spirochaetes  by  being  extremely  steep,  or,  in  other  words,  by 

11  Lustgarten,  Wien.  med.  Woch.,  xxxiv,  1884. 

"Schaudinn  und  Hoffmann,  Arb.  a.  d.  kais.  Gesundheitsamt,  22,  1905. 


DISEASES  CAUSED   BY   SPIROCH^TES  851 

forming  acute,  rather  than  obtuse,  angles.  The  ends  of  the  micro- 
organism are  delicately  tapering -and  come  to  a  point.  In  his  iirst 
investigations,  Schaudimi  was  unable  to  discover  flagella  and 
believed  that  he  saw  a  marginal  undulating  membrane  similar  to  that 
noticed  in  the  trypanosomes.  Later  observations  by  this  observer, 
as  well  as  by  others,  revealed  a  delicate  flagellum  at  each  end,  but 
left  the  existence  of  an  undulating  membrane  in  doubt.  Uncertain, 
in  his  later  investigations,  whether  the  microorganisms  described 
by  him  could  scientifically  be  classified  with  the  spirochaste  proper, 
Schaudinn  suggested  the  name  of  " Treponema  pallidum." 

In  the  same  preparations  in  which  Spirochaeta  pallida  was  first 
seen,  other  spirochaetes  were  present,  which  were  easily  distinguished 
from  the  former  by  their  coarser  con- 
tours, their  flatter  and  fewer  undula- 
tions, their  more  highly  refractile  cell 
bodies,  and,  in  stained  preparations, 
their  deeper  color.  These  microorgan- 
isms were  not  found  regularly,  and 
were  interpreted  merely  as  fortuitous 
and  unimportant  companions.  To  them 
Schaudinn  gave  the  name  of  "Spiro- 

cha3ta  refringens."  FlG-    OI.-SPIBOCHBTA    PAL- 

ml  -,  .         -I  .  ,.      LIDA.     Spleen,  congenital  syph- 

The  epoch-making  d  i  s  c  o  v  e  r  y  of  /T       ,./• 

ihs.     (Levaditi  method.) 

bchaudmn    and    Hoffmann    was    soon 

confirmed  by  many  observers,  and  the  etiological  relationship 
of  Spirochaeta  pallida  to  syphilis  may  now  be  regarded  as  an  accepted 
fact.  Although  our  inability  to  cultivate  the  microorganism  has 
made  it  impossible  to  carry  out  Koch's  postulates,  nevertheless  in- 
direct evidence  of  such  a  convincing  nature  has  accumulated  that 
no  reasonable  doubt  as  to  its  causative  importance  can  be  retained. 
The  spirochaetes  have  been  found  constantly  present  in  the  primary 
and  secondary  lesions  of  all  carefully  investigated  cases,  and,  so  far, 
have  invariably  been  absent  in  subjects  not  afflicted  with  syphilis. 

Schaudinn  himself,  not  long  after  his  original  communication, 
was  able  to  report  seventy  cases  of  primary  and  secondary  syphilis 
in  which  these  microorganisms  were  found.  Spitzer12  found  them 
constantly  present  in  a  large  number  of  similar  cases.  Sobernheim 
and  Tomasczewski14  found  the  spirochagtes  in  fifty  cases  of  primary 

"Spitzer,  Wien.  klin.  Woch.,  1905. 

14  Sobernheim  und  Tomasczewski,  Munch,  med.  Woch.,  1905. 


852  PATHOGENIC   MICROORGANISMS 

and  secondary  syphilis,  but  failed  to  find  them  in  eight  tertiary 
cases.  Mulzer,15  who  found  the  microorganisms  invariably  in  twenty 
cases  of  clinical  syphilis,  failed  to  find  them  in  fifty-six  carefully 
investigated  non-syphilitic  subjects.  The  voluminous  confirmatory 
literature  which  has  accumulated  upon  the  subject  can  not  here  he 
reviewed.  The  presence  of  these  spirochaetes  in  the  blood  at  certain 
stages  of  the  disease  has  been  demonstrated  by  Bandi  and  Simonelli16 
who  found  them  in  the  blood  taken  from  the  roseola  spots,  and  by 
Lcvaditi  and  Petresco17  who  found  them  in  the  fluid  of  blisters 
produced  upon  the  skin. 

In  tertiary  lesions  the  spirochaetes  have  been  found  less  regularly 
than  in  the  primary  and  secondary  lesions,  but  positive  evidence 
of  their  presence  has  been  brought  by  Tomasczewski/8  Ewing,19 
and  others  who  succeeded  in  demonstrating  them  in  gummata. 


FIG.  92. — SPIROCH^JTA  PALLIDA,     Liver,  congenital  syphilis.     (Levaditi  method.) 

Noguehi  and  Moore20  have  recently  found  the  Spirochaeta  pallida 
in  the  brain  of  patients  dead  of  general  paresis. 

In  congenital  syphilis,  many  observers  have  found  Spirochaeta 
pallida  in  the  lungs,  liver,  spleen,  pancreas,  and  kidneys,  and,  in 
isolated  cases,  in  the  heart  muscle.  The  organisms  were  always 
present  in  large  numbers  and  practically  in  pure  culture.  These 
results  more  than  any  others  seem  to  furnish  positive  proof  of  the 
etiological  relationship  between  the  spirochaete  and  the  disease. 

Demonstration  of  Treponema  pallidum. — In  the  living  state  the 
spirochaetes  have  been  observed  in  the  hanging  drop  or  under  a 
coverslip  rimmed  with  vaseline.  It  is  extremely  important,  in  pre- 

15  Mulzer,  Berl.  klin.  Woch.,  1905,  and  Archiv  f .  Dermat.  u.  Syph.,  79,  1906. 

16  Bandi  und  Simonelli,  Cent.  f.  Bakt.,  40,  1905. 

17  Levaditi  et  Petresco,  Presse  med.,   1905. 

18  Tomasczewski,  Munch,  med.  Woch.,  1906. 

18  Ewing,  Proc.  N.  Y.  Path.  Soe.,  N.  S.,  5,  1905. 
20  Noguehi  and  Moore,  Jour.  Exp.  Med.,  xvii,  1913. 


DISEASES  CAUSED   BY  SPOROCH^TES  853 

paring  such  specimens  from  primary  lesions  or  from  lymph  glands, 
to  obtain  the  material  from  the  deeper  tissues,  and  thus  as  uncon- 
taminated  as  possible  by  the  secondary  infecting  agents  present 
upon  the  surface  of  an  ulcer,  and  also  as  free  from  blood  as  possible. 
It  is  best  to  employ  a  special  device  known  as  a  "condenser  for  dark- 
field  illumination "  (Dunkel-Kammer-Beleuchtung).  This  apparatus  is 
screwed  into  the  place  of  the  Abbe  condenser.  The  preparation  is 
made  upon  a  slide  and  covered  with  a  cover-slip  as  usual.  A  drop 
of  oil  is  then  placed  upon  the  upper  surface  of  the  condenser 
and  the  slide  laid  upon  it  so  that  an  even  layer  of  oil,  without  air- 
bubbles,  intervenes  between  the  top  of  the  dark  chamber  and  the 
bottom  of  the  slide.  An  arc  light  furnishes  the  most  favorable 
illumination.  In  such  preparations  the  highly  refractive  cell-bodies 
stand  out  against  the  black  background,  and  the  motility  of  the 
organisms  may  be  observed.21 

The  dark-field  condenser  is  without  question  the  easiest  method 
of  finding  the  Spirochaeta  pallida.  Its  use  is  easily  learned  and  the 
apparatus  is  sufficiently  cheap  so  that  it  lends  itself  to  the  use 
of  the  clinic  and  the  office.  With  very  little  practice  it  is  possible 
to  detect  the  spirochaete  in  suspension  if  care  is  taken  that  not  too 
much  blood  or  other  solid  particles  are  mixed  with  the  preparation. 
Should  it  be  impossible  to  obtain  the  material  scraped  from  syphilitic 
lesions  in  a  sufficiently  dilute  condition  it  is  best  to  emulsify  it  in 
a  drop  or  two  of  human  ascitic  fluid. 

EXAMINATION  IN  SMEARS. — The  Spirochaeta  pallida  can  not  be 
stained  with  the  weaker  anilin  dyes,  and  even  more  powerful  dyes, 
such  as  carbol-fuchsin  and  gentian-violet,  give  but  a  pale  and  un- 
satisfactory preparation.  The  staining  method  most  commonly  used 
is  the  one  originally  recommended  by  Schaudinn  and  Hoffmann. 
This  depends  upon  the  use  of  Giemsa's  azur-eosin  stain  employed  in 
various  modifications.  The  most  satisfactory  method  of  applying 
this  solution  is  as  follows: 

Make  smears  upon  slides  or  cover-slips,  if  possible  from  the  depth  of  the 
lesions,  'as  free  as  possible  from  blood. 

Fix  in  methyl  alcohol  for  ten  to  twenty  minutes  and  dry. 

Cover  the  preparation  with  a  solution  freshly  prepared  as  follows: 

Distilled  water  10  c.c. 

Potassium  carbonate  1 : 1,00 .  .  0 5-10  gtt. 

21  For  a  critical  summary  of  the  various  methods  of  dark-field  illumination,  the 
reader  is  referred  to  an  article  by  Siedentopf,  Zeit.  f.  wiss.  Mikrosc.,  xxv,  1908. 


854  PATHOGENIC   MICROORGANISMS 

Add  to  this: 

Giemsa 's  solution  (fur  EomanowsTci  Farbung) 10-12  gtt. 

This  staining  fluid  is  left  on  for  one  to  four  hours,  preferably  in  a  moist 
chamber.  Wash  in  running  water.  Blot. 

By  this  method  Spirochaeta  pallida  is  stained  characteristically 
with  a  violet  or  reddish  tinge. 

A  rapid  and  convenient  method  for  staining  such  smears  consists 
in  the  use  of  azur  I  and  eosin  in  aqueous  solutions  as  recommended 
by  Wood.  The  smears  are  fixed  in  methyl  alcohol  as  before  and 
are  then  flooded  with  the  azur  I  solution.  The  eosin  solution  is  then 
dropped  on  the  preparation  until  an  iridescent  pellicle  begins 
to  form.  Satisfactory  preparations  may  be  obtained  by  this  method 
after  ten  or  fifteen  minutes  of  staining. 

A  fairly  satisfactory  method  of  staining  the  treponema  pallidum  in 
smear-preparations  is  that  of  Fontana.22  For  this  method,  the  fol- 
lowing solutions  are  necessary : 

1.  Acetic  acid  1  c.c. 

Formalin    2  c.c. 

Distilled  water 100  c.c. 

Leave  in  one  minute;  wash  in  water. 

2.  Phenol  86  per  cent   (liquefied  crystals) 1  c.c. 

Tannic  acid    ...  * 5  grams 

Distilled  water  83  c.c. 

Cover  preparation  with  this  and  steam  gently  one-half  minute;  wash. 

3.  Silver   nitrate    0.25  gram 

Distilled  water 100  c.c. 

Ammonia  q.  s. 

Add  ammonia  drop  by  drop  until  the  precipitate  which  first  appears  goes 
into  solution.  Steam  one-half  minute;  wash. 

Recently  a  rapid  and  extremely  simple  but  not  very  reliable 
method  for  the  demonstration  of  Spirochaeta  pallida  in  smears,  by 
the  use  of  India  ink,  has  been  described. 

Smears  are  prepared  in  the  following  way:  A  drop  of  the  fluid 
squeezed  out  of  the  syphilitic  lesion,  as  free  as  possible  from  blood 
cells,  is  mixed,  on  a  slide,  with  a  drop  of  India  ink  (best  variety 
is  "Chin  chin"  Giinther-Wagner  Liquid  Pearl  ink),  and  the  mixture 


22  See  Levaditi  and  BanfcowsTci:  Ann  de  1'Inst.  Past.,  1913,  XXVII,  p.  583. 


DISEASES  CAUSED   BY  SPIROCH^TES  855 

smeared  with  the  edge  of  another  slide  as  in  making  blood  smears. 
When  the  smear  dries,  which  takes  about  a  minute,  it  may  be 
immediately  examined  with  an  oil-immersion  lens.  The  organisms 
are  seen  unstained  on  a  black  background. 

DEMONSTRATION  OF  SPIROCH^TES  IN  TISSUES. — Ordinary  histo- 
logical  staining  methods  do  not  reveal  the  spirochaetes  in  tissue 
sections.  It  is  customary,  therefore,  to  employ  some  modification  of 
Cajal's  silver  impregnation.  The  technique  most  commonly  em- 
ployed is  that  known  as  Levaditi's  method,2*  which  is  carried  out  as 
follows : 

The  fresh  tissue  is  cut  into  small  pieces  which  should  not  be  thicker  than 
2  to  4  millimeters. 

Fix  in  10%  formalin  (4%  formaldehyde)  for  twenty- four  hours.  Wash 
in  water. 

Dehydrate  in  96%  alcohol  twenty-four  hours.     Wash  in  water. 

Place  in  a  3%  silver-nitrate  solution  at  incubator  temperature  (37.5°  C.) 
and  in  the  dark  for  3  to  5  days.  Wash  in  water  for  a  short  time. 

Place  in  the  following  solution   (freshly  prepared) : 

Pyrogallic   acid    2-4  grams. 

Formalin    5  c.c. 

Distilled  water 100     " 

Leave  in  this  for  twenty-four  to  forty-eight  hours  at  room  temperature. 
Wash  in  water. 
Dehydrate  in  graded  alcohols. 
Embed  in  paraffin  and  cut  thin  sections. 

The  sections  may  be  examined  without  further  staining,  or,  if  desired, 
may  be  weakly  counterstained  with  Giemsa's  solution  or  hematoxylin. 

A  modification  of  this  method  which  has  been  much  recommended 
is  that  of  Levaditi  and  Manouelian.'2*  The  directions  given  by  these 
authors  are  as  follows : 

Fix  in  formalin  as  in  previous  method. 

Dehydrate  in  96%  alcohol  twelve  to  twenty-four  hours.  Wash  in  distilled 
water. 

Place  in  a  1%  silver-nitrate  solution  to  which  10%  of  pyridin  has  been 
added  just  before  use. 

Leave  in  this  solution  for  two  to  three  hours  at  room  temperature  and 
from  four  to  six  hours  at  50°  C.  approximately. 

Wash  rapidly  in  10%  pyridin. 

23  Levaditi,  Comptes  rend,  de  la  soc.  de  biol.,  59,  1905. 

24  Levaditi  et  Manouelian,  Comptes  rend,  de  la  soc.  de  biol.,  60,  1906. 


856  PATHOGENIC   MICROORGANISMS 

Place  in  a  solution  containing  4%  of  pyrogallic  acid  to  which  10%  of 
C.  P.  acetone,  and  15%  (per  volume)  of  pyridin  have  been  added  just  before 
use.  Leave  in  this  solution  two  to  three  hours. 

Wash  in  water,  dehydrate  in  graded  alcohols,  and  embed  in  paraffin  by 
the  usual  technique. 

Examined  after  treatment  by  either  of  these  methods,  the  spiro- 
ehaetes  appear  as  black,  untransparent  bodies  lying  chiefly  extra- 
cellularly.  They  are  characteristically  massed  about  the  blood- 
vessels of  the  organs  and  only  exceptionally  seem  to  penetrate  into 
the  interior  of  the  parenchyma  cells. 

Attempts  at  cultivating  Spirochaeta  pallida  were  at  first  unsuc- 
cessful. In  1909  Schereschewsky25  reported  that  he  had  suc- 
ceeded in  obtaining  multiplication  of  the  organisms  on  artificial 
media  as  follows :  Sterile  horse  serum  in  centrifuge  tubes  was  coagu- 
lated at  60°  C.  until  it  assumed  a  jelly-like  consistency.  It  was  then 
placed  in  the  incubator  at  37.5°  C.  for  three  days  before  being  used. 
The  cultures  were  planted  by  snipping  off  a  small  piece  of  tissue 
from  a  syphilitic  lesion,  dropping  it  into  such  a  tube,  and  causing 
it  to  sink  to  the  bottom  by  means  of  centrifugalization.  The  tube 
was  then  tightly  stoppered  with  a  cork.  In  such  anaerobic  serum 
cultures  Schereschewsky  claims  to  have  grown  the  organisms  for 
several  generations,  though  not  in  pure  culture. 

Miihlens  also  obtained  growth  of  Spirochaeta  pallida  in  horse 
serum  agar  by  a  method  which  is  very  similar  to  that  of  Schereschew- 
sky. The  most  extensive  and  convincing  work  on  treponema  palli- 
dum  has  been  more  recently  by  Noguchi.  Noguchi26  began  his  work 
in  1910  and  1911.  His  first  successful  cultivations  were  made  from 
the  syphilis-infected  testicles  of  rabbits,  and  after  many  unsuccessful 
attempts,  with  slightly  varying  media  and  technique,  he  finally  suc- 
ceeded in  the  following  way:  He  prepared  tubes  (20  cm.  high  and 
1.5  cm.  wide),  containing  10  c.c.  of  a  serum-water  made  of  distilled 
water,  three  parts;  and  horse,  sheep,  or  rabbit  serum,  one  part. 
These  were  sterilized  by  the  fractional  method  in  the  usual  way 
(15  minutes  each  day).  Into  them  was  then  placed  a  small  piece 
of  sterile  rabbit  kidney  or  testicle  and  a  bit  of  the  testicle  of  a 
syphilitic  rabbit,  in  which  many  spirochaetes  were  present.  The 
fluid  was  then  covered  with  sterile  paraffin  oil  and  placed  in  an 


26  Schereschewsky,  Deut.  med.  Woch.,  N.   S.,  xix  and  xxix,  1909. 
28  Noguchi   Jour.  Exp.  Med.,  xiv,  1911;  xvii,  1913. 


DISEASES   CAUSED   BY   SPIROCH.ETES  857 

anaerobic  jar.  After  ten  days  at  33.5°  C.  the  spirochaetes  had 
multiplied  considerably,  in  all  but  one  case,  together  with  bacteria. 
He  obtained  pure  cultures  from  these  initial  cultivations  after  much 
difficulty,  by  a  number  of  methods.  At  first  he  succeeded  only  by 
allowing  the  spirochgetes  to  grow  through  Berkefeld  filters,  which 
they  did  on  the  fifth  day.  A  better  method  more  recently  adopted 
by  him  consists  in  preparing  high  tubes  of  three  parts  of  very 
slightly  alkaline  or  neutral  agar  to  which  a  piece  of  sterile  tissue 
has  been  added.  These  tubes  are  then  inoculated  from  the  impure 
cultures  with  a  long  pipette.  Close  to  the  tissue  and  along  the 
stab  the  spirochaetes  and  bacteria  will  grow  and,  after  about  ten 
days  to  two  weeks,  the  spirochastes  will  have  wandered  away  from 
the  stab  and  will  be  visible  as  hazy  colonies.  They  can  then  be 
fished,  after  cutting  the  tubes,  and  directly  transplanted  to  other 
serum-a gar-tissue  tubes  prepared  as  before,  and  eventually  will 
grow  in  pure  culture.  By  this  method  Noguchi  has  also  cultivated 
pure  cultures  from  lesions  in  monkeys. 

The  writer,  with  Hopkins,  has  successfully  applied  Noguchi 's 
method  and  has  found  that,  after  once  cultivated  artificially,,  the 
treponema  pallidum  can  be  obtained  in  quantity  best  by  cultivation 
in  flasks  containing  heated  or  unheated  rabbit  kidney  with  ascitic 
broth  and  sealed  with  paraffin.  Recently  we  have  been  using  modifi- 
cations of  a  method  worked  out  in  our  laboratory  by  Miss  Gilbert, 
in  which  slanted  egg,  with  or  without  glycerin,  made  as  for  tubercle 
cultivation,  is  used  instead  of  kidney  tissue.  This  is  put  up  in  high 
tubes  and  ascitic  broth  and  paraffin  oil  added.  By  this  method, 
large  quantities  of  culture  pallida  are  obtained  within  two  weeks 
and  can  be  concentrated  in  large  quantities. 

Animal  Pathogenicity. — Until  very  recently,  all  experimental 
inoculation  of  animals  was  unsuccessful.  During  the  year  1903 
Metchnikoff  and  Roux27  finally  succeeded  in  transmitting  the  disease 
to  monkeys.  The  monkey  first  used  by  these  observers  was  a  female 
chimpanzee.  At  the  point  of  inoculation,  the  clitoris,  there  appeared, 
twenty-six  days  after  inoculation,  a  typical  indurated  chancre,  which 
was  soon  followed  by  swelling  of  the  inguinal  glands.  Fifty-six 
days  after  the  inoculation  there  appeared  a  typical  secondary  erup- 
tion, together  with  swelling  of  the  spleen  and  of  the  lymph  nodes. 
Similar  successful  experiments  were  made  soon  after  this  by  Lassar.28 

"Metchnikoff  et  Eoux,  Ann.  de  1'inst.  Pasteur,  1903,  1904,  and  1905. 
28  Lassar,  Berl.  klin.  Woch.,  xl,  1903. 


858  PATHOGENIC   MICROORGANISMS 

Soon  after  the  experiments  of  Metchnikoff  and  Roux,  successful 
inoculations  upon  lower  monkeys  (macacus)  were  carried  out  by 
Nicolle.29  Since  that  time,  it  has  been  found  by  various  observers 
that  almost  all  species  of  monkeys  are  susceptible.  Simple  sub- 
cutaneous injection  is  not  sufficient  to  produce  a  lesion.  The  tech- 
nique which  has  given  the  most  satisfactory  results  consists  in  the 
cutaneous  implantation  of  small  quantities  of  syphilitic  tissue  ob- 
tained by  excision  or  curetting  of  primary  and  secondary  lesions.  A 
small  pocket  is  made  under  the  mucous  membrane  of  the  genitals 
or  of  the  'eyebrows  and  the  tissue  placed  in  this  under  aseptic 
precautions.  The  inoculation  may  be  made  directly  from  the  human 
being,  but  can  also  be  successfully  carried  out  from  monkey  to 
monkey  for  many  generations.  Attempts  at  transmission  from  ter- 
tiary lesions  have  so  far  been  unsuccessful.  The  spirochaetes  can 
be  demonstrated  both  in  the  primary  lesions  of  the  inoculated  animal 
and  in  the  secondarily  enlarged  glands.  The  successful  inoculation 
of  rabbits  with  syphilis  has  been  recently  performed  by  Bertarelli.30 
He  obtained  ulcerative  lesions  by  inoculation  upon  the  cornea  and 
into,  the  anterior  chamber  of  the  eye  and  was  able  to  prove  the 
syphilitic  nature  of  these  lesions  by  finding  the  spirochaete  within 
the  tissue.  In  these  animals,  as  well  as  in  the  lower  monkeys,  the 
disease  usually  remains  localized. 

In  1907  Parodi  showed  that  syphilitic  lesions  could  be  produced 
by  direct  inoculation  into  the  testicles  of  rabbits.  This  method  of 
inoculation  has  been  subsequently  studied  by  many  investigators, 
especially  by  Uhlenhuth  and  Mulzer.31  It  is  the  easiest  method  of 
obtaining  spirochaete  in  any  quantity  from  lesions  in  man.  The 
spirochaate-containing  lesions  may  be  either  excised  or  scraped  as 
conditions  permit  and  rubbed  up  in  a  mortar  with  sterile  sand, 
in  a  few  centimeters  of  sterile  human  ascitic  fluid.  This  emulsion 
is  then  injected  directly  into  the  substance  of  rabbit  testicles.  A 
swelling  supervenes  which  is  often  noticeable  after  two  weeks,  and 
is  usually  at  its  height  in  five  to  seven  weeks.  At  this  time  the 
testicle  is  much  larger  than  normal,  sometimes  evenly  swollen  and 
sometimes  nodular,  and  of  a  firm  elastic  consistency.  When  taken 
out  at  castration  it  oo/es  a  sticky  fluid,  both  from  testicle  and 
tunica,  which  is  rich  in  actively  motile  spirochaetes.  By  continuous 


29  Nicollr,  Ann.  do  1'inst.  Pasteur,  1903. 

30  Bertarelli,  Cent.   f.  Bakt.,  xli,   1906. 

n  Uhlenhuth  und  Mulzer,  Arb.  a.  d.  k.  Gesimdh't's  Amt.,  xxxiii,  1909. 


DISEASES  CAUSED   BY  SPIROCH^TES  859 

transinoenlation  from  one  rabbit  to  another  such  a  strain  can  be 
indefinitely  carried  along.  It  can  be  inoculated  from  rabbits  to 
monkeys  and  vice  versa.  This  method  as  well  as  Noguchi's  cultiva- 
tions have  opened  a  new  era  of  spirochaete  investigation.  It  is 
stated  by  some  observers  that  intravenous  inoculation  of  rabbits 
may  be  followed  by  localization  in  the  testis  and  occasionally  gum- 
matous  infections  in  other  parts  of  the  body  have  been  induced 
after  such  inoculation  by  Uhlenhuth,  Mulzer,  and  others.  Brown 
and  Pearce  in  an  elaborate  series  of  recent  investigations  published 
in  Journal  of  Experimental  Medicine  in  1920,  have  succeeded  in 
reproducing  almost  all  types  of  syphilitic  lesions  in  rabbits  by 
appropriate  methods  of  inoculation. 

Immunization  in  Syphilis. — It  is  a  well-known  fact  observed  by 
clinicians  that  during  active  syphilis  the  patient  cannot  be  superin- 
fected.  That  this  resistance  develops  quite  rapidly  was  shown  by 
Metchnikoff  and  Roux,  who  found  that  reinfection  of  a  monkey  was 
possible  if  attempted  within  two  weeks  of  the  first  inoculation,  but 
was  unsuccessful  if  delayed  beyond  this  period. 

On  the  basis  of  this  knowledge,  Metchnikoff,32  Finger  and  Land- 
steiner,33  and  others  have  made  attempts  to  devise  some  method  of 
immunization.  They  attempted  to  attenuate  the  syphilitic  virus 
by  repeated  passage  through  monkeys.  These  experiments  were 
unsuccessful,  the  last-mentioned  observers  finding  absolutely  no  at- 
tenuation after  twelve  generations  of  monkey  inoculation. 

Bertarelli  and  others  have  shown  that  the  production  of  a 
syphilitic  lesion  on  the  cornea  of  one  eye  does  not  protect  against 
an  inoculation  done  on  the  other.  Rabbits  that  have  been  inoculated 
with  spirochaete  material  and  that  have  not  developed  syphilitic  dis- 
ease can  be  successfully  inoculated  on  subsequent  attempts.  The 
offspring  of  female  rabbits  with  syphilis  of  the  cornea  are,  according 
to  Muhlens,  not  immune. 

There  is  no  evidence  so  far  that  specific  therapy  or  treatment 
with  spirochaete  material  has  had  favorable  influence  upon  the  disease. 
Chemotherapy  has  had  results  analogous  to  those  obtained  in  man.34 

Attempts  at  passive  immunization  have  been  entirely  without 
success. 

8*  Metchnikoff,  Arch.  gen.  de  m6d.,  1905. 

33  Finger  und  Landsteiner,  Sitzungsber.  d.  Wien.  Akad.  d.  Wiss.,  1905. 

34  Von  Prowazelc,  "Handbuch  der  pathogenen  Protozoen,"  i,    1912,   Leipzig, 
Bartsch. 


860  PATHOGENIC   MICROORGANISMS 

Investigations  carried  on  in  our  own  laboratory*  in  the  last  three 
years  have  shown  definitely,  we  think,  that  immunization  of  animals 
with  culture  pallida  produces  antibodies,  agglutinins,  treponemacidal 
substances,  entirely  analogous  to  similar  substances  produced  against 
bacteria.  However,  there  is  a  biological  change  which  takes  place 
when  treponema  pallidum  is  cultivated.  The  antibodies  produced 
with  the  culture  pallida  have  no  action  whatsoever  upon  the  virulent 
organisms.  The  latter,  indeed,  seem  to  be  entirely  insulated  against 
such  antibodies  and  do  not  induce  antibody  formation  to  any  great 
extent,  in  either  the  infected  animal  or  man.  Both  active  and 
passive  immunization  with  culture  pallida  and  the  sera  produced 
with  them  have  no  effect.  We  have  obtained  some  evidence,  how- 
ever, that  in  rabbits  a  purely  local  resistance  develops  in  the  tissue 
previously  the  site  of  a  lesion. 

The  occurrence  of  a  Wassermann  reaction  was  formerly  supposed 
to  indicate  the  existence  of  specific  syphilitic  antibodies  in  the  serum 
of  patients.  More  recent  information  regarding  this  reaction  seems 
to  show  that  it  depends  upon  the  presence  in  the  serum  of  syphilitic 
patients  of  substances  produced  indirectly  because  of  the  presence 
of  syphilitic  infection.  It  may  be  a  relative  increase  of  globulins 
or,  as  Schmidt  has  suggested,  a  change  in  the  physical  state  of  the 
globulins  or  other  substances  present  in  the  serum.  At  any  rate 
it  has  been  found  that  the  fixation  of  complement  in  the  Wasser- 
mann reaction  does  not  depend  upon  the  occurrence  of  a  specific 
antigen-antibody  reaction.  In  the  first  place  the  antigens  most  com- 
monly used,  and  successfully  so,  in  the  Wassermann  reactions,  are 
non-specific  lipoidal  extracts  of  normal  organs. 

Again  it  has  been  demonstrated  that  extracts  of  cultures  of  the 
Spirochaeta  pallida  as  well  as  extractions  from  the  testes  of  syphilitic 
rabbits  do  not  furnish  an  antigen  suitable  for  the  Wassermann 
reaction.  This  has  followed  especially  from  the  work  of  Noguchi,35 
of  Craig  and  Nichols,36  and  ourselves.  This  forms  a  corollary  to 
the  other  experiments  previously  mentioned  and  shows  that,  what- 
ever the  Wassermann  reaction  may  be,  it  is  not  a  specific  comple- 
ment fixation  in  the  sense  of  Bordet  and  Gengou.  It  must  be 
admitted,  therefore,  that  our  knowledge  of  syphilis  immunity  is  in 


*  Zinsser  and  Hopkins,  Series  of  papers,  Journ.  Exp.  Med.,  1915  and  1916. 

85  Noguchi,  Jour.  Am.  Med.  Assoc.,  1912. 

36  Craig  and  Nichols,  Jour.  Exp.  Med.,  xvi,  1912. 


DISEASES  CAUSED  BY  SPIROCILETES  861 

its  infancy  and  that  we  know  very  little  about  the  systemic  reactions 
which  follow  infection  with  the  Spirochaeta  pallida. 

The  fact  that  the  syphilitic  virus  does  not  pass  through  a  filter 
has  been  demonstrated  by  Klingmiiller  and  Baermann,37  who  in- 
oculated themselves  with  filtrates  from  syphilitic  material. 

Hopkins  and  the  writer  have  carried  out  some  seventy  filtration 
experiments  with  culture  treponemata  at  various  stages  of  growth 
without  ever  obtaining  filter  passage.  It  is  our  opinion  that  the 
assumption  of  a  filtrable  stage  of  the  syphilitic  virus  is  entirely  un- 
justified and  devoid  of  valid  experimental  support. 


THE  SPIRQCHJETES  OF  RELAPSING  FEVER 

The  microorganisms  causing  relapsing  fever  were  first  observed 
in  1873,  by  Obermeier,38  who  demonstrated  them  in  the  blood  of 
patients  suffering  from  this  distinct  type-  of  fever.  Since  his  time 
extensive  studies  by  many  other  observers  have  proven  beyond 
question  the  etiological  connection  between  the  disease  and  the 
organisms. 

Morphology  and  Staining. — The  spirochaste  of  Obermeier  is  a 
delicate  spiral  thread  measuring  from  7  to  9  micra  in  length  (Novy), 
and  about  1  micron  in  thickness.  While  this  is 'its  average  size,  it 
may,  according  to  some  observers,  be  considerably  longer  than  this, 
its  undulations  varying  from  four  to  ten  or  more  in  number.  Com- 
pared with  the  red  blood  cells  among  which  they  are  seen,  the 
microorganisms  may  vary  from  one-half  to  nine  or  ten  times  the 
diameter  of  a  corpuscle.  In  fresh  preparations  of  the  blood,  very 
active  corkscrew-like  motility  and  definite  lateral  oscillation  are 
observed.  In  stained  preparations  no  definite  cellular  structure  can 
be  made  out,  the  cell  body  appearing  homogeneous,  except  in  de- 
generated individuals,  in  which  irregular  granulation  or  beading 
has  been  observed.  Flagella  have  been  described  by  various  ob- 
servers. Novy  and  Knapp39  believe  that  the  organisms  possess  only 
one  terminal  flagellum.  Zettnow,40  on  the  other  hand,  claims  to 
have  demonstrated  lateral  flagella  by  special  methods  of  staining. 


37  Klingmuller  und  Baermann,  Dent.  med.  Woch.,  1904. 

38  Obermeier,  Cent.  f.  d.  med.  Wiss.,  11,  1873. 

39  Novy  and  Knapp,  Jour,  of  Infec.  Dis.,  3,  1906. 
"Zettnow,  Deut.  med.  Woch.,  32,  1906. 


862 


PATHOGENIC   MICROORGANISMS 


Norris,  Pappenheimer,  and  Flournoy,41  in  smears  stained  by  poly- 
chrome methods,  have  described  long,  filamentous  tapering  ends 
which  they  interpreted  as  bipolar,  terminal  flagella,  never  observing 
more  than  one  at  each  end.  Spores  are  not  found. 

Cultivation. — Innumerable  attempts  to  induce  these  microorgan- 
isms to  multiply  upon  artificial  media  have  been  made.    Novy  and 


FIG.  93. — SPIROCILETE  OF  RELAPSING  FEVER.     (After  Norris.  Pappenheimer,  and 

Flournoy.) 

Knapp  succeeded  in  keeping  the  microorganisms  alive  and  virulent 
in  the  original  blood  for  as  long  as  forty  days,  and  call  attention 
to  the  fact  that  the  length  of  time  for  which  they  may  be  kept  alive 
depends  to  a  great  extent  upon  the  stage  of  fever  at  which  the 
blood  is  removed  from  the  patient.  They  do  not,  however,  believe 
that  extensive  multiplication,  or,  in  other  words,  actual  cultivation, 
had  taken  place  in  their  experiments.  Norris,  Pappenheimer,  and 
Flournoy,  on  the  other  hand,  have  obtained  positive  evidence  of 


"Norris,  Pappenheimer ,  and  Flournoy,  Joiir.  of  Inf.  Dis.,  3,   1906. 


DISEASES  CAUSED  BY  SPIROCH^TES 


863 


multiplication  of  the  spirochaetes  in  fluid  media.  They  obtained 
their  cultures  by  inoculating  a  few  drops  of  spirochsetal  rat  blood 
into  3  to  5  c.c.  of  citrated  human  or  rat  blood.  Smears  made  from 
these  tubes,  after  preservation  for  twenty-four  hours  at  room  tem- 
perature, showed  the  microorganisms  in  greater  number  than  in  the 
original  infected  blood.  A  similar  multiplication  could  be  observed 
in  transfers  made  from  these  "first-generation"  tubes  to  other  tubes 
of  citrated  blood.  Attempts  at  cultivation  for  a  third  generation, 
however,  failed. 

Noguchi42  has  lately  successfully  cultivated  the  spirochaete  of 


FIG.  94. — SPIROCH^TE  OF  RELAPSING  FEVER.     Citrated  normal  rat  blood. 
Norris,  Pappenheimer,  and  Flournoy.) 


(After 


Obermeier  in  ascitic  fluid  containing  a  piece  of  sterile  rabbit 's  kidney 
and  a  few  drops  of  citrated  blood  under  anaerobic  conditions. 

Four  different,  probably  distinct  varieties  of  spirochaete  have 
been  described  in  connection  with  relapsing  fever,  all  of  which  have 
been  cultivated  by  Noguchi  by  means  of  this  method.  The  first  is 
known  as  the  spirochaete  of  Obermeier  mentioned  above.  Probably 
distinct  are  the  Spirochaeta  Duttoni  of  West  African  Tick  fever 
described  by  Button  and  Todd43  in  1905,  the  Spirochaeta  Kochi,  and 

42  Noguchi,  Jour.  Exp.  Med.,  xvii,  1913. 
"Button  and  Todd,  Brit.  Med.  Jour.,  1905. 


864  PATHOGENIC   MICROORGANISMS 

the  Spirochseta  Novyi,44  the  organism  studied  by  Norris  and  Flour- 
noy  and  Pappenheimer,  and  regarded  as  a  different  species  by  them. 

Pathogenicity. — Inoculation  with  blood  containing  these  spiro- 
chaetes produces  disease  in  monkeys,  rats,  and  mice.  Attempts  to 
transmit  the  disease  experimentally  to  dogs,  rabbits,  and  guinea-pigs 
have  so  far  been  unsuccessful.  The  subcutaneous  inoculation  of 
monkeys  is  followed  after  from  two  to  four  days  by  a  rise  of 
temperature  which  occurs  abruptly  as  is  the  case  in  the  disease 
in  man  and  which  may  last  several  days.  During  this  time  the 
spirochaetes  can  be  found  in  the  blood  of  the  animals  just  as  it  is 
found  in  that  of  infected  human  beings.  The  temperature  subsides 
after  a  day  or  more,  when  it  again  rapidly  returns  to  normal.  As 
a  rule,  the  paroxysms  are  not  repeated.  Occasionally,  however,  two 
or  three  attacks  may  supervene  before  immunity  is  established.  In 
rats,  an  incubation  time  of  from  two  to  five  days  occurs.  At  the 
end  of  this  time  the  spirochaetes  may  be  found  in  large  numbers 
in  the  blood,  and  the  animals  show  symptoms  of  a  severe  systemic 
infection.  The  attack  lasts  from  four  to  five  days,  at  the  end 
of  which  time  the  microorganisms  again  disappear.  Occasionally 
even  in  these  animals  relapses  have  been  observed.  Gross  patholog- 
ical changes  are  not  found,  with  the  exception  of  an  enlargement 
of  the  spleen. 

In  man  the  disease  caused  by  the  spirochaete  of  Obermeier  and 
allied  organisms  commonly  known  as  relapsing  fever,  is  common 
in  Eastern  Europe,  India,  Africa,  and  most  of  the  warmer  countries. 
It  has,  from  time  to  time,  been  observed  epidemically  in  Europe, 
especially  in  Eussia,  and  a  few  epidemics  have  occurred  in  the 
Southern  United  States.  The  disease  comes  on  abruptly,  beginning 
usually  with  a  chill  accompanied  by  a  sharp  rise  of  temperature 
and  generalized  pains.  Together  with  the  rise  of  temperature,  which 
often  exceeds  104°  F.,  there  are  great  prostration  and  occasionally 
delirium.  Early  in  the  disease  the  spleen  becomes  palpable  and 
jaundice  may  appear.  The  spirochaetes  are  easily  detected  in  the 
blood  during  the  persistence  of  the  fever,  w^hich  lasts  usually  from 
three  to  ten  days.  At  the  end  of  this  time  the  temperature  usually 
drops  as  suddenly  as  it  rose,  and  the  general  symptoms  rapidly 
disappear.  After  a  free  interval  of  from  one  to  three  weeks  a  relapse 
may  occur,  which  is  usually  less  severe  and  of  shorter  duration  than 

**Novy  and  Fraenlcel,  cited  from  Noguchi. 


DISEASES   CAUSED   BY  SPIROCH^TES 


865 


the  original  attack.  Two,  three,  or  even  four  attacks  may  occur, 
but  the  disease  is  not  very  often  fatal.  When  patients  do  succumb, 
however,  the  autopsy  findings  are  not  particularly  characteristic. 
Apart  from  the  marked  enlargement  of  the  spleen,  which  histologic- 
ally  shows  the  changes  indicating  simple  hyperplasia,  and  a  slight 
enlargement  of  the  liver,  no  lesions  are  found.  The  diagnosis  is 
easily  made  during  the  febrile  stage  by  examination  of  a  small  quan- 
tity of  blood  under  a  cover-slip  or  in  the  hanging-drop  preparation. 


FIG.  95. — SPIROCHAETE  OF  RELAPSING  FEVER.     (From  preparation  furnished  by 

Dr.  G.  N.  Calkins.) 


Several  types  of  relapsing  fever  have  been  described.  In  Africa 
the  disease  has  long  been  prevalent  in  many  regions  and  the  in- 
vestigations of  Ross  and  Milne,45  Koch,46  Button  and  Todd,47  and 
others  have  brought  to  light  that  many  conditions  occurring  among 
the  natives,  formerly  regarded  as  malarial,  are  caused  by  a  species 
of  spirochaete.  Whether  or  not  the  microorganisms  observed  in  the 
African  disease  are  exactly  identical  with  the  spirochaete  observed 
by  Obermeier  is  yet  a  question  about  which  several  opinions  are 
held.  Dutton  and  Todd  believe  that  the  same  microorganism  is 
responsible  for  both  diseases.  Koch,  on  the  other  hand,  believes 
that  the  slightly  smaller  size  of  the  African  spirochaete  and  the 
milder  course  of  the  clinical  symptoms  indicate  a  definite  difference 

"Ross  and  Milne,  Brit.  Med.  Jour.,  1904. 

44  Koch,  Deut.  mod.  Woch.,  xxxi,  1905. 

"Dutton  and  Todd,  Lancet,  1905,  and  Jour,  of  Trop.  Med.,  1905. 


866 


PATHOGENIC   MICROORGANISMS 


between  the  two.  Animal  experiments  made  with  the  African  or- 
ganism, furthermore,  usually  show  a  much  more  severe  infection 
than  do  similar  inoculations  with  the  European  variety.  The  spiro- 
chaete  found  in  the  African  disease  is  usually  spoken  of  at  present 
as  ' '  Spirochaeta  Duttoni."  Novy  and  Knapp,48.  after  extensive 
studies  with  the  microorganisms  from  various  sources,  have  come 
to  the  conclusion  that,  although  closely  related,  definite  species 
differences  exist  between  the  two  types  mentioned  above,  and  that 
these  again  are  definitely  distinguished  from  similar  organisms 
described  by  Turnbull49  as  occurring  in  a  similar  disease  observed 
in  India. 

The  mode  of  transmission  of  this  disease  is  not  clear  for  all  types. 


:: 


FIG.  96.-^SpiROCHvETE   OF  DUTTON,   AFRICAN  TICK  FEVER.     (From  preparation 
furnished  by  Dr.  G.  N.  Calkins.) 


Dutton  and  Todd,  however,  were  able  to  show  satisfactorily  that, 
in  the  case  of  the  African  disease  at  least,  transmission  occurs 
through  the  intermediation  of  a  species  of  tick.  The  conditions 
under  which  such  intermediation  occurs  have  been  carefully  studied 
by  Koch.50  The  tick  (Ornithodorus  moubata)  infects  itself  when 
sucking  blood  from  an  infected  human  being.  The  spirochaete  may 
remain  alive  and  demonstrable  within  the  body  of  the  tick  for 
as  long  as  three  days.  Koch  has  shown,  furthermore,  that  they 
may  be  found  also  within  the  eggs  laid  by  an  infected  female  tick. 
He  succeeded  in  producing  experimental  infection  in  monkeys  by 


48  Novy  and  Knapp,  loc.  cit. 
"Turnlm!!,  Indian  Mcd.  Gaz.,  1905. 
50  Koch,  Berl.  med.  Woch.,  1906. 


DISEASES   CAUSED   BY   SPIROCH^TES  867 

subjecting  the  animals  to  the  bites  of  the  infected  insects.  For  the 
European  variety  of  the  disease  no  such  intermediate  host  has  as  yet 
been  demonstrated  with  absolute  certainty.  It  is  known,  however, 
that  the  organism  can  live  in  the  bodies  of  bed  bugs  and  it  has  also 
been  suggested  that  lice  may  be  the  carriers.  Lice  also  are  regarded 
as  the  transmitting  agent  of  a  similar  relapsing  fever  prevalent  in 
North  Africa  caused  by  the  Spiroschaudinnia,  berberi. 

Immunity. — It  has  long  been  a  well-known  fact  that  recovery 
from  an  attack  of  relapsing  fever  usually  results  in  a  more  or  less 
definite  immunity.  The  blood  of  human  beings,  monkeys,  and  rats 
which  have  recovered  from  an  attack  of  this  disease  show  definite 
and  specific  bactericidal  and  agglutinating  substances,  and  Novy 
and  Knapp  have  demonstrated  that  the  blood  serum  of  such  animals 
may  be  used  to  confer  passive  immunity  upon  others. 

VINCENT'S    ANGINA 

The  condition  known  as  Vincent's  angina  consists  of  an  inflam- 
matory lesion  in  the  mouth,  pharynx,  or  throat,  situated  most  fre- 
quently upon  the  tonsils.  The  disease  usually  begins  as  an  acute 
stomatitis,  pharyngitis,  or  tonsillitis,  which  soon  leads  to  the  forma- 
tion of  a  pseudo-membrane,  which,  at  this  stage,  has  a  great  deal 
of  resemblance  to  that  caused  by  the  diphtheria  bacillus.  At  later 
stages  of  the  disease  there  may  be  distinct  ulceration,  the  ulcers 
having  a  well-defined  margin  and  "punched-out"  appearance,  so 
that  clinically  they  have  often  been  erroneously  diagnosed  as 
syphilis.  Apart  from  the  localized  pain,  the  disease  is  usually  mild, 
but  occasionally  moderate  fever  and  systemic  disturbances  have 
been  observed.  Unlike  diphtheria  and  syphilis,  this  peculiar  form 
of  angina  usually  yields,  without  difficulty,  to  local  treatment. 

The  nature  of  lesions  of  this  peculiar  kind  was  not  clear  until 
Plaut,51  Vincent,52  and  others  reported  uniform  bacteriological  find- 
ings in  cases  of  this  description.  These  observers  have  been  able 
to  demonstrate  in  smears  from  the  lesions  a  spindle-shaped  or  fusi- 
form bacillus,  together  with  which  there  is  usually  found  a  spirillum 
not  unlike  the  spirillum  of  relapsing  fever.  The  two  microorganisms 
are  almost  always  found  together  in  tliis  form  of  disease  and  were 


r>lPlaul,   IViit.  mod.  Wocli.,  xlix,  LS04. 

•"-  rincait,   Ann.   do   1'mst.   Pasteur,   389(5,   and    Bull,   et   mem.    do   la  sue.   mod. 
des  hop.  de  P.,   1898. 


868 


PATHOGENIC   MICROORGANISMS 


regarded  by  the  first  observers  as  representing  two  distinct  forms 
dwelling  in  symbiosis. 

The  fusiform  bacilli  described  by  Vincent,  Plaut,  Babes,  and  others, 
are  from  3  to  10  micra  in  length,  and  have  a  thickness  at  the  center 
varying  from  0.5  to  0.8  micron.  From  the  center  they  taper  grad- 
ually toward  the  ends,  ending  in  blunt  or  sharp  points.  The  length 
of  these  bacilli  may  vary  greatly  within  one  and  the  same  smear 
preparation.  They  are  usually  straight,  sometimes  slightly  curved. 
They  do  not  stain  very  easily  with  the  weaker  anilin  dyes,  but  are 
readily  stained  by  Loeffler's  methylene-blue,  carbol-fuchsin,  or  bet- 


FIG.  97. — THROAT  SMEAR,  VINCENT'S  ANGINA.     Fusiform  bacilli  and  spirilla. 

ter,  by  Giemsa's  stain.  Stained  by  Gram,  they  are  usually  de- 
colorized, though  in  this  respect  the  writers  have  found  them  to 
vary.  Stained  preparations  show  a  characteristic  inequality  in  the 
intensity  of  the  stain,  the  bacilli  being  more  deeply  stained  near 
the  end,  and  showing  a  banded  or  striped  alternation  of  stained 
and  unstained  areas  in  the  central  body.  Their  staining  qualities 
in  this  respect  are  not  unlike  those  of  the  diphtheria  bacillus,  and 
according  to  Babes53  the  dark  areas  are  to  be  interpreted  as  meta- 
chromatic  granules.  The  bacilli  are  not  motile. 

The  spirilla  found  in  Vincent's  angina  are  usually  somewhat 
longer  than  the  fusiform  bacilli,  and  are  made  up  of  a  variable 
number  of  undulations,  shallow  and  irregular  in  their  curvatures, 


88  Babes,  in  Kolle  und  Wassermann,  1.  Erganzungsband,  1907, 


DISEASES  CAUSED  BY  SPOROCH^TES  869 

unlike  the  more  regularly  steep  waves  of  Spirochaeta  pallida.  They 
are  stained  with  even  more  difficulty  than  are  the  bacilli  and  usually 
appear  less  distinct  in  the  preparations.  The  stain,  however,  is 
taken  without  irregularity,  showing  none  of  the  metachromatism  ob- 
served in  the  bacilli. 

By  the  earlier  observers  cultivation  of  these  microorganisms  was 
attempted  without  success.  Recently,  however,  it  has  been  shown 
that  cultivation  could  be  carried  out  under  anaerobic  conditions. 
Tunnicliff54  has  cultivated  the  organisms  anaerobically  upon  slants 
of  ascitic  agar  at  37.5°  C.  This  observer  found  that  in  such  cultures, 
before  the  fifth  day,  bacilli  only  could  be  found,  that  after  this 
time,  however,  spirilla  gradually  appeared  and  finally  constituted 
the  majority  of  the  organisms  in  the  culture.  It  appeared  to  Tun- 
nicliff from  this  study  that  the  spirilla  might  be  developed  out  of 
the  fusiform  microorganisms  representing  the  adult  form.  This, 
howevr,  is  an  error. 

The  microorganisms  of  Vincent's  angina,  when  occurring  in  the 
throat,  are  rarely  present  alone,  being  usually  accompanied  by  other 
microorganisms,  such  as  staphylococci,  streptococci,  and  not  infre- 
quently diphtheria  bacilli.  When  occurring  together  with  diph- 
theria, they  are  said,  by  some  German  observers,  to  aggravate  the 
latter  condition  considerably.  This  frequent  association  with  other 
microorganisms  renders  it  impossible  to  decide  conclusively  that  the 
fusiform  bacilli  and  spirilla  are  the  primary  etiological  factors  in 
these  inflammations.  It  has  been  frequently  suggested  that  they 
may  be  present  as  secondary  invaders  upon  the  soil  prepared  for 
them  by  other  microorganisms. 

Animal  inoculation  with  these  microorganisms  has  led  to  little 
result. 

Fusiform  Bacilli  other  than  those  in  Vincent's  Angina. — Fusiform 
bacilli  morphologically  indistinguishable  from  those  found  in  the  angina  of 
Vincent  may  frequently  be  found  in  smears  taken  from  the  gums,  from 
carious  teeth,  and  occasionally  among  the  microorganisms  in  the  pus  from 
old  sinuses.  Several  varieties  of  these  bacilli  have  been  described  in  con- 
nection with  definite  pathological  conditions. 

Babes,55  in  1893,  observed  spindle-shaped  bacilli  not  unlike  those  described 
above,  but  somewhat  shorter,  in  histological  sections  prepared  from  tissues 
from  the  gums  of  individuals  suffering  from  scurvy.  He  found  similar  bacilli 

54  Tunnicliff ',  Jour,  of  Infec.  Dis.,  3,  1906. 
56  Babes,  Deut.  med.  Woch.,  xliii,  1893. 


870  PATHOGENIC   MICROORGANISMS 

in  rabbits  intravenously  inoculated  with  material  from  the  patients  and  was 
ab'le  to  cultivate  the  bacilli  for  sevei  al  generations.  His  descriptions,  however, 
of  the  microorganisms  as  found  in  the  secondary  cultures  vary  considerably 
from  those  of  the  original  findings  in  the.  gums  of  the  patients.  His  results 
are  not  convincing. 

In  uoma,  a  gangrenous  disease  of  the  gums  and  cheeks,  occurring  occa- 
sionally in  individuals  who  have  been  severely  run  down  by  acute  infectious 
diseases  or  great  hardship,  Weaver  and  Tunnicliff  have  found  spirilla  and 
fusiform  bacilli  in  large  numbers.  The  organisms  were  present  not  only 
in  smears  from  the  surface,  but  were  also  found  by  histological  methods, 
in  large  numbers,  lying  in  the  tissues  beyond  the  area  of  necrosis.  Here 
again  it  is  not  entirely  certain  whether  these  microorganisms  were  the  primary 
etiological  factors  or  whether  they  are  to  be  regarded  merely  as  secondary 
invaders  of  a  necrotic  focus. 

Fusiform  bacilli  are  cultivated  with  greater  ease  than  formerly  supposed; 
we  have  found  it  relatively  simple  to  grow  them  together  with  Gram  positive 
cocci  in  symbiosis  in  simple  broth  tubes  covered  with  paraffin  oil  without 
the  addition  of  any  enriching  substance  and  in  similar  symbiotic  conditions 
on  infusion  agar  plates  under  incomplete  anaerobic  conditions.  In  such  plates 
they  form  curious  colonies  in  which  the  fusiform  bacilli  and  micrococci  are 
intimately  commingled.  Krumwiede56  has  had  no  difficulty  in  cultivating 
them  in  pure  culture  in  anaerobic  plates. 

SPIROCHJETA    PERTENUE 

In  a  disease  known  as  ' t  Framboesia  tropica,"  or  popularly 
"Yaws,"  occurring  in  tropical  and  subtropical  countries  and  much 
resembling  syphilis,  Castellani,57  in  1905,  was  able  to  demonstrate 
a  species  of  spirochaete  which  has  a  close  morphological  resemblance 
to  Spirochseta  pallida.  The  microorganism  was  found  in  a  large 
percentage  of  the  cases  examined  both  in  the  cutaneous  papules  and 
in  ulcerations.  Confirmatory  investigations  on  a  larger  series  of 
cases  were  later  carried  out  by  von  dem  Borne.58 

The  microorganism  is  from  7  to  20  micra  in  length  with  numerous 
undulations  and  pointed  ends.  Examined  in  fresh  preparations,  it 
has  an  active  motility  similar  to  that  of  Spirochasta  pallida.  In 
smears  it  is  easily  stained  by  means  of  the  Giemsa  method. 

Both  the  clinical  similarity  between  yaws  and  syphilis,  as  well 
as  the  similarity  between  the  microorganisms  causing  the  diseases, 

58  Krumwiede,  Jour.  Inf.  Dis.,   1913. 

"  Castellani,  Brit.  Med.  Jour.,  1905,  and  Deut.  med.  Woch.,  1906. 

Kvon  dem  Borne,  Jour.  Trop.  Med.,  10,  1907. 


DISEASES  CAUSED  BY  SPIROCH^TES  871 

has  opened  the  question  as  to  the  identity  of  the  two  microorganisms. 
According  to  most  clinical  observers,  however,  yaws,  which  is  a 
disease  characterized  chiefly  by  a  generalized  papular  eruption,  is 
unquestionably  distinct,  clinically,  from  lues,  and  experiments  of 
Neisser,  Baermann,  and  Halberstadter,59  as  well  as  of  Castellani60 
himself,  have  tended  to  show  that  there  is  a  distinct  difference 
between  the  immunity  produced  by  attacks  of  the  two  diseases. 
The  disease  is  transmissible  to  monkeys,  as  is  syphilis. 

SPIROCH^TA    GALLINARUM 

An  acute  infectious  disease  occurring  among  chickens,  chiefly  in 
South  America,  has  been  shown  by  Marchoux  and  Salimbeni61  to  be 
caused  by  a  spirochaete  which  has  much  morphological  similarity  to 
the  spirochaete  of  Obermeier. 

The  disease  comes  on  rather  suddenly  with  fever,  diarrhea,  and 
great  exhaustion,  and  often  ends  fatally.  The  spirochaete  is  easily 
demonstrated  in  the  circulating  blood  of  the  animals  by  staining 
blood-smears  with  Giemsa's  stain  or  with  dilute  carbol-fuchsin. 

Artificial  cultivation  of  the  microorganism  has  not  yet  been 
accomplished.  Experimental  transmission  from  animal  to  animal 
is  easily  carried  out  by  the  subcutaneous  injection  of  blood.  Other 
birds,  such  as  geese,  ducks,  and  pigeons,  are  susceptible;  mammals 
have,  so  far,  not  been  successfully  inoculated.  According  to  the 
investigation  of  Leviditi  and  Manouelian,62  the  spirochaates  are 
found  not  only  in  the  blood  but  thickly  distributed  throughout  the 
various  organs. 

Under  natural  conditions,  infection  of  chickens  seems  to  depend 
upon  a  species  of  tick  which  acts  as  an  intermediate  host  and  causes 
infection  by  its  bite.  The  spirochaete,  according  to  Marchoux  and 
Salimbeni,  may  be  found  in  the  intestinal  canal  of  the  ticks  for 
as  long  as  five  months  after  their  infection  from  a  diseased  fowl. 

In  the  blood  of  animals  which  have  survived  an  infection,  ag- 
glutinating substances  appear  and  active  immunization  of  animals 
may  be  carried  out  by  the  injection  of  infected  blood  in  which  the 
spirochaetes  havo  boon  killed,  either  by  moderate  heat  or  by  preserva- 


09  Neisser,  Baermann,  und  Halberstadter,  Miinch.  mod.  Wodi.,  xxviii,  1906. 

60  Castellani,  Jour,  of  Hyg.,  7,  1907. 

61  Marchoux  et  Salimbeni,  Ann.  de  Tinst..  Pasteur,  1903. 
K  Levaditi  et  Manouelian,  Ann.  de  Pinst.  Pasteur,  1906. 


872 


PATHOGENIC   MICROORGANISMS 


tion  at  room  temperature.    The  serum  of  immune  animals,  further- 
more, has  a  protective  action  upon  other  birds. 

It  is  not  impossible  that  the  Spirochaeta  gallinarum  may  be 
identical  with  the  Spirochaeta  anserina  previously  discovered  by 
Sacharoff.63  This  last-named  microorganism  causes  a  disease  in 
geese,  observed  especially  in  Eussia  and  Northern  Africa,  which  both 
clinically  and  in  its  pathological  lesions  corresponds  closely  to  the 
disease  above  described  as  occurring  in  chickens.  The  spirochaete 
is  found  during  the  febrile  period  of  the  disease  in  the  circulating 
blood,  is  morphologically  indistinguishable  from  the  spirochaete  of 


FIG.  98-  -SPIROCH^ETE  GALLINARUM.     (From  preparation  furnished  by  Dr.  G.  N. 

Calkins.) 

chickens,  and  can  not  be  cultivated  artificially.  The  similarity  is 
further  strengthened  by  the  fact  that  Spirochaeta  anserina  is 
pathogenic  for  other  birds,  but  not  for  animals  of  other  genera. 
Noguchi  has  succeeded  in  cultivating  Spirochaeta  gallinarum  by  the 
same  method  by  which  he  has  cultivated  the  organisms  of  relapsing 
fever.  Ascitic  fluid  tubes  with  a  piece  of  sterile  rabbit  kidney 
were  inoculated  with  a  few  drops  of  blood  containing  the  spirochaetes 
and  cultivated  at  37.5°  C.  under  anaerobic  conditions. 

Spirochaeta  phagedenis. — This  is  an  organism  cultivated  by 
Noguchi  by  his  ascitic-fluid-tissue  method  from  phagedenic  lesions 
on  human  external  genitals.  It  is  probably  a  new  species. 


Sacharoff,  Ann.  de  1'inst.  Pasteur,  1891. 


DISEASES  CAUSED   BY  SPIROCH.ETES  873 

Spirochaeta  macrodentium. — Cultivated  by  Noguchi  ;64  is  believed 
by  him  to  be  identical  with  the  spirochaete  found  in  Vincent 's  angina. 

Spirochaeta  microdentium. — A  similar  organism  with  wide  con- 
volutions, cultivated  by  Noguchi  from  the  tooth  deposits  chiefly  in 
children.  It  was  grown  on  mixtures  of  sheep  serum  water  and 
sterile  tissue  in  a  way  similar  to  that  employed  by  him  for  other 
organisms  of  this  group. 

Spirochaeta  calligynun, — Cultivated  by  Noguchi65  from  condy- 
lomata — is  probably  a  new  species. 

Rat-Bite  Fever. — Rat-bite  fever  is  a  peculiar  disease,  which, 
after  an  incubation  period  of  ten  or  more  days,  is  characterized 
by  fever,  headache  and  inflammation  at  the  site  of  the  bite,  swollen 
lymph  glands,  skin  eruption  and  pains.  After  three  to  six  days 
the  fever  ceases  and  an  afebrile  period  of  two  or  three  days  ensues.66 
After  this  the  fever  again  occurs.  Recently  Futaki,  Takaki,  Taniguchi, 
and  Osumi67  have  described  a  treponema  which  they  have  called 
Treponema  morsus  muris.  It  is  a  spiral  organism,  somewhat  larger 
than  the  Treponema  pallidum,  and  is  found  in  the  skin,  the  lymph 
nodes,  and  in  the  blood.  They  have  succeeded  in  inoculating  rats 
and  have  cultivated  it  in  Schimamine  medium,  which  consists  of 
100  c.c.  of  horse  serum  in  which  0.5  to  0.75  gram  of  sodium  nucleate 
is  dissolved  and  carbon  dioxide  passed  through  the  solution  until 
the  serum  becomes  transparent.  It  is  then  heated  for  three  days 
at  60°,  and  on  the  fourth  day  at  65°  until  it  coagulates.  This 
medium  is  deeply  inoculated,  but  no  other  anaerobic  precautions  are 
taken. 

"Noguchi,  Jour.  Exp.  Med.,  xv,   1912. 

65  Noguchi,  Jour.  Exp.  Med.,  xvii,  1913. 

66  In    connection   with   Eat-Bite   Fever   see    also    Kaneko   and   Okuda,   Journal 
Exp.  Med.,  vol.  xxvi,  1917,  p.  363. 

67  Jour.  Exp.  Med.,  1916,  xxiii,  p.  249. 


CHAPTER   XLIV 

YELLOW  FEVER  AND  THE  LEPTOSPIRA  ICTEROIDES,  WEIL'S  DIS- 
EASE (INFECTIOUS  JAUNDICE)  AND  LEPTROSPIRA  ICTERO- 
HEMORRILEGLZE 

YELLOW  fever  is  an  acute  infectious  disease  which  prevails  en- 
demically  in  the  tropical  countries  of  the  Western  Hemisphere,  but 
occurs  also  along  the  western  coast  of  Africa  and  has  exceptionally 
appeared,  in  epidemic  invasons,  in  the  north  temperate  United 
States  and  Europe.  Guiteras,  as  quoted  by  Osier,  classifies  the 
distribution  of  the  disease  into  three  areas  of  infection. 

1.  The    area   in  which  the   disease   is  never   absent,   including 
tropical  South  American  ports  and  Havana. 

2.  The  area   of  periodic  epidemics,  including  sea-ports  of  the 
tropical  Atlantic  in  America  and  Africa. 

3.  The  area  of  accidental  epidemics,  extending  from  parallel  45° 
north  latitude  to  35°  south  latitude.     In  the  United  States  severe 
epidemics  have  frequently  occurred  in  Louisiana,  Mississippi,  and 
Alabama,  and  occasional  but  severe   epidemics  have   occurred  in 
Philadelphia  and  Baltimore. 

The  disease  occurs  spontaneously  only  in  man,  and  experimental 
inoculation  of  lower  animals  has  been  successful  only  in  the  chim- 
panzee in  a  single  case  reported  by  Thomas.1 

In  man  afflicted  with  the  malady  the  clinical  picture  is  one  of 
a  rapidly  developing  fever  with  severe  gastrointestinal  symptoms, 
vomiting  of  blood,  albuminuria,  and  often  active  delirium.  The  mor- 
tality is  usually  high,  often  reaching  eighty  per  cent  or  more  in 
the  severe  epidemics. 

Mode  of  Transmission. — Until  comparatively  recent  years  the 
mode  of  transmission  of  yellow  fever  was  not  understood  and  many 
erroneous  theories  were  prevalent.  It  was  supposed  that  yellow 
fever  was  contagious,  and  transmitted  from  person  to  person  by 
direct  or  indirect  contact  with  those  afflicted  or  by  fomites.  The 
first  to  make  the  definite  assertion  that  yellow  fever  was  transmitted 

1  Thomas,  Brit.  Med.  Jour.,  1,  1907. 

874 


YELLOW  FEVER  AND  THE  LEPTOSPIRA  875 

by  the  agency  of  mosquitoes  was  Carlos  Finlay.  Finlay,2  as  early 
as  188.1,  advanced  the  theory  that  mosquitoes  were  responsible  for 
the  transmission  of  this  disease  and,  furthermore,  recognized  "Ste- 
gomyia  fasciata"  or  "Stegomyia  calopus"  as  the  guilty  species. 
Finlay 's  opinion,  although  later  proved  to  be  correct,  was  at  first 
based  only  upon  such  circumstantial  evidence  as  the  correspondence 
of  the  yellow-fever  zones  with  the  distribution  of  this  species  of 
mosquito  and  the  great  prevalence  of  mosquitoes  at  times  during 
which  epidemics  occurred.  His  theory  was,  therefore,  received  with 
much  skepticism  and  was  neglected  by  scientists  until  its  revival  in 
1900,  when  the  problem  was  extensively  investigated  by  a  com- 
mission of  American  army  surgeons. 

Reed,  Carroll,  Agramonte,  and  Lazear  were  the  members  of  this 
commission.  The  courage,  self-sacrifice,  and  scientific  accuracy 
which  characterized  the  work  of  these  men  have  made  the  chapter 
of  yellow  fever  one  of  the  most  brilliant  in  the  annals  of  American 
scientific  achievement. 

Their  work  was  much  facilitated  by  the  experience  of  Gorgas3 
and  others,  who  had  demonstrated  the  absolute  failure  of  ordinary 
sanitary  regulations  to  limit  the  spread  of  yellow  fever. 

They  began  their  researches  by  investigating  carefully  the 
validity  of  Sanarelli's  claims  as  to  the  etiological  significance  of 
his  "Bacillus  icteroides. "  The  results  of  this  work  yielded  ab- 
solutely no  basis  for  confirmation. 

They  then  proceeded  to  investigate  the  possibility  of  an  inter- 
mediate host. 

In  August,  1900,  the  commission  began  its  work  on  this  subject 
by  allowing  mosquitoes,4  chiefly  those  of  the  stegomyia  species,  to 
suck  blood  from  patients,  later  causing  the  same  insects  to  feed 
upon  normal  susceptible  individuals.  The  first  nine  experiments 
were  negative.  The  tenth,  of  wrhich  Carroll  was  the  subject,  was 
successful.  Four  days  after  being  bitten  by  the  infected  insect 
Carroll  became  severely  ill  with  an  attack  of  yellow  fever,  by  which 


2  Finlay,  Ann.  Roy.  Acad.  d.  Havana,   1881. 

'Gorgas,  Jour,  of  Trop.  Med.,  1903. 

*Eeed,  Carroll,  Agramonte,  and  Lazear,  Phila.  Med.  Jour.,  Oct.,  1900;  also 
Am.  Pub.  Health  Assn.  Rep.,  1903;  Agramonte,  N.  Y.  Med.  News,  1900;  Reed, 
Jour,  of  Hyg.,  1902;  Reed,  Carroll,  and  Auramontc,  Am.  Medicine,  July,  1901. 
Boston  Med.  and  Surg.  Jour.,  14,  1901;  Carroll,  Jour.  Am.  Med.  Assn.,  40,  1903; 
Carrol,  ' '  Yellow  Fever ' '  in  Mense,  ' '  Handbuch  der  Tropen-Krankheiten, ' '  ii. 


876  PATHOGENIC   MICROORGANISMS 

his  life  was  endangered,  and  from  the  effects  of  which  he  died  several 
years  later. 

On  the  13th  of  September,  Lazear,  while  working  in  the  yellow- 
fever  wards,  noticed  that  a  stegomyia  had  settled  upon  his  hand, 
and  deliberately  allowed  the  insect  to  drink  its  fill.  Five  days  later 
he  became  ill  with  yellow  fever  and  died  after  a  violent  and  short 
illness. 

With  these  experiences  as  a  working  basis,  the  commission  now 
decided  upon  a  more  systematic  and  thoroughly  controlled  plan  of 
experimentation. 

In  November  of  the  same  year,  1900,  an  experiment  station, 
"Camp  Lazear,"  was  established  in  the  neighborhood  of  Havana, 
about  a  mile  from  the  town  of  Quemados.  The  camp  was  surrounded 
by  the  strictest  quarantine.  Volunteers  from  the  army  of  occupation 
were  called  for,  and  twelve  individuals  were  selected  for  the  camp, 
three  immunes  and  nine  non-immunes.  Two  of  the  latter  were 
physicians.  The  immunes  and  the  members  of  the  commission  only 
were  allowed  to  go  in  and  out.  All  non-immunes  who  left  the 
camp  were  prohibited  from  re-entering  and  their  places  taken  by 
other  non-immune  volunteers.  During  December,  five  of  the  non- 
immune  inmates  were  successfully  inoculated  with  yellow  fever  by 
means  of  infected  mosquitoes.  During  January  and  February  five 
further  successful  experiments  were  made.  Clinical  observations 
were  made  by  experienced  native  physicians,  Carlos  Finlay  among 
them,  and  the  patients,  as  soon  as  they  were  unquestionably  ill 
with  yellow  fever,  were  removed  to  a  yellow-fever  hospital.  This 
was  done  to  prevent  the  possibility  of  the  disease  spreading  within 
the  camp  itself.  The  mosquitoes  used  for  the  experiments  were 
all  cultivated  from  the  larva  and  kept  at  a  temperature  of  about 
26.5°  C. 

A  further  important  experiment  was  now  made.  A  small  house 
was  erected  and  fitted  with  absolutely  mosquito-proof  windows  and 
doors.  The  interior  was  divided  by  wire  mosquito  netting  into  two 
spaces.  Within  one  of  these  spaces  fifteen  infected  mosquitoes  were 
liberated.  Seven  of  these  had  fed  upon  yellow-fever  patients  four 
days  previously;  four,  eight  days  previously;  three,  twelve  days 
previously ;  and  one,  twenty-four  hours  previously.  A  non-immune 
person  then  entered  this  room  and  remained  there  about  thirty 
minutes,  allowing  lumself  to  be  bitten  by  seven  mosquitoes.  Twice 
after  this  the  same  person  entered  the  room,  remaining  in  it  alto- 


YELLOW  FEVER  AND  THE  LEPTOSPIRA  877 

gether  sixty-four  minutes  and  being  bitten  fifteen  times.  After 
four  days  this  individual  came  down  with  yellow  fever. 

In  the  other  room  two  non-immunes  slept  for  thirteen  nights 
without  any  evil  results  whatever. 

It  now  remained  to  show  that  mosquitoes  were  the  sole  means 
of  transmission  and  to  exclude  the  possibility  of  infection  by  contact 
with  excreta,  vomitus,  or  fomites.  For  this  purpose  another  mos- 
quito-proof house  was  constructed.  By  artificial  heating  its  tem- 
perature was  kept  above  32.2°  C.  and  the  air  was  kept  moist  by 
the  evaporation  of  water.  Clothing  and  bedding,  vessels,  and  eating 
utensils,  soiled  with  vomitus,  blood,  and  feces  of  yellow-fever 
patients  were  placed  in  this  house  and  three  non-immune  persons 
inhabited  it  for  twenty  days.  During  this  time  they  were  strictly 
quarantined  and  protected  from  mosquitoes.  Each  evening,  before 
going  to  bed,  they  unpacked  and  thoroughly  shook  clothing  and 
bedding  of  yellow-fever  patients,  and  hung  and  scattered  these 
materials  about  their  beds.  They  slept,  moreover,  in  contact  with 
linen  and  blankets  soiled  by  patients.  None  of  these  persons  con- 
tracted yellow  fever.  The  same  experiment  was  twice  repeated  by 
other  non-immunes,  in  both  cases  with  like  negative  results. 

All  of  the  non-immunes  taking  part  in  these  experiments  were 
American  soldiers.  Four  of  them  were  later  shown  to  be  susceptible 
to  yellow  fever  by  the  agencies  of  mosquito  infection  or  blood- 
injection. 

The  results  obtained  by  the  investigations  of  this  commission 
may  be  summarized,  therefore,  as  follows: 

Yellow  fever  is  acquired  spontaneously  only  by  the  bite  of  the 
Stegomyia  fasciata.  It  is  necessary  that  the  infecting  insect  shall 
have  sucked  the  blood  of  a  yellow-fever  patient  during  the  first 
four  or  five  days  of  the  disease,  and  that  an  interval  of  at  least 
twelve  days  shall  have  elapsed  between  the  sucking  of  blood  and 
the  reinfection  of  another  human  being.  Sucking  of  the  blood  of 
patients  advanced  beyond  the  fifth  day  of  the  disease  does  not  seem 
to  render  the  mosquito  infectious,  and  at  least  twelve  days  are 
apparently  required  to  allow  the  parasite  to  develop  within  the 
infected  mosquito  to  a  stage  at  which  reinfection  of  the  human 
being  is  possible. 

The  results  of  the  American  Commission  were  soon  confirmed  by 
Guiteras5  and  by  Marchoux,  Salimbeni,  and  Simond.6  These  latter 

5  Guiteras,  Eev.  d.  med.  trop.  Jan.,  1901,  and  Am.  Med.,  11,  1901. 
*  Marchoux,  Salimbeni,  and  Simond,  Ann.   de  Tinst.  Pasteur,   1903. 


878  PATHOGENIC   MICROORGANISMS 

observers,  moreover,  confirmed  the  fact  that  infection  could  be 
experimentally  produced  by  injections  of  blood  or  blood  serum  taken 
from  patients  during  the  first  three  days  of  the  disease.  They 
showed  that  blood  taken  after  the  fourth  day  was  no  longer  in- 
fectious: that  0.1  c.c.  of  serum  sufficed  for  infection  and  finally 
that  no  infection  could  take  place  through  excoriations  upon  the 
skin.  They  furthermore  confirmed  the  observation  of  Carroll  that 
the  virus  of  the  disease  could  pass  through  the  coarser  Berkefcld 
and  Chamberland  filters, — passing  through  a  Chamberland  candle 
"F"  but  held  back  by  the  finer  variety  known  as  "B." 

The  fundamental  factors  of  yellow-fever  transmission  thus  dis- 
covered, we  are  in  possession  of  logical  means  of  defense.  The  most 
important  feature  of  such  preventive  measures  must  naturally  center 
upon  the  extermination  of  the  transmitting  species  of  mosquito. 

Stegomyia  fasciata  or  calopus  is  a  member  of  the  group  of 
' '  Culicidae. "  It  is  more  delicately  built  than  most  of  the  other 
members  of  the  group  culicidae,  is  of  a  dark  gray  color,  and  has 
peculiar  thorax-markings  which  serve  to  distinguish  it  from  other 
species.  The  more  detailed  points  of  differentiation  upon  which 
an  exact  zoological  recognition  depends  are  too  technical  to  be 
entered  into  at  this  place.  Briefly  described,  they  consist  of  lyre-like 
markings  of  the  back,  unspotted  wings,  white  stripes  and  spots  on 
the  abdomen,  and  bandlike  white  markings  about  the  metatarsi  and 
tarsi  of  the  third  pair  of  legs.  The  peculiar  power  of  transmitting 
yellow  fever  possessed  by  this  species  is  explained  by  Marchoux 
and  Simond7  by  the  fact  that  Stegomyia  fasciata  is  unique  among 
culicidae  in  that  the  female  lives  for  prolonged  periods  after  sucking 
blood.  Among  other  species — Culex  fatigens,  Culex  confirmatus,  and 
most  others — the  female  lays  its  eggs  within  from  two  to  eight  days 
after  feeding  on  blood  and  rarely  lives  longer  than  the  twelfth  day 
—the  time  necessary  for  the  development  of  the  yellow-fever 
parasite. 

The  limitation  of  yellow  fever  to  tropical  countries8  is  explained 
by  the  fact  that  stegomyia  develops  only  in  places  where  high 
temperatures  prevail.  The  optimum  temperature  for  this  species 
lies  between  26°  and  32°  C.  At  17°  C.  it  no  longer  feeds,  and 
bcomes  practically  paralyzed  at  15°  C.  In  order  to  thrive,  the 


''Marchoux  and  Simond,  Ann.  de  1'inst.  Pasteur,  1906. 

8  Otto,  in   Kolle   und   Wassermann,    "Handbuch,"   etc.,    11,   Erganzungsband. 


YELLOW  FEVER  AND  THE   LEPTOSPIRA 


879 


species  requires  a  temperature  never  going  below  22°  C.  at  night 
and  rising  regularly  above  25°  C.  during  the  day.  The  females  only 
are  dangerous  as  sources  of  infection.  The  insect,  like  Anopheles, 
has  the  peculiarity  of  feeding  chiefly  at  night. 


FIG.  99. — STEGOMYIA  FASCIATA.     (a)  Female.     (6)  Male.     (After  Carroll.) 

Experiments  done  by  Reed,  Carroll,  Agramonte,  and  Lazear,  to 
ascertain  whether  the  power  of  infecting  was  hereditarily  transmis- 
sible from  the  mosquito  to  following  generations,  were  negative.  A 
positive  result,  however,  has  been  reported  by  Marchoux  and 
Simond.9  This  question  must  still  await  more  extensive  research. 


ETIOLOGY    OF    YELLOW    FEVER 

Numerous  researches  have  been  aimed  at  the  elucidation  of  the 
problem  of  etiology  and  a  large  number  of  different  microorganisms 
for  which  etiological  significance  was  claimed,  have  been  isolated 
from  dejecta,  vomitus  and  secretions  of  patients.  A  few  of  these 
claims  have  only  historical  importance,  but  may  be  mentioned  be- 
cause of  the  wide  interest  they  aroused  among  bacteriologists  in  the 
past. 


9  Marchoux  and  Simond,  Comptes  rend,  de  la  soc.  de  biol.,  59,  1905. 


880  PATHOGENIC   MICROORGANISMS 

Cornil  and  Babes,10  in  1883,  described  chained  cocci  to  which 
they  attributed  etiological  significance,  but  their  contentions  have 
remained  entirely  unconfirmed.  Sternberg,11  in  1897,  described  a 
colon-like  organism,  "bacillus  X,"  for  which  he  made  very  con- 
servative claims,  which  he  himself,  later,  withdrew. 

The  most  active  discussion  was  roused  by  the  announcement  of 
Sanarelli,12  in  1897,  that  he  had  discovered,  in  the  blood  and  tissues 
of  patients  dead  of  yellow  fever,  a  Gram-negative  bacillus,  which 
he  believed  to  be  the  etiological  agent  of  the  disease.  He  based  his 
contention  upon  the  facts  that  he  had  isolated  the  organism  from 
seven  cases  of  yellow  fever,  had  produced  symptoms  similar  to  the 
disease  of  the  human  being  by  the  inoculation  of  pure  cultures  into 
dogs,  and  had  obtained  agglutination  of  the  bacillus  in  the  serum 
of  convalescent  patients.  Later  he  inoculated  five  human  beings 
subcutaneously  with  sterilized  cultures  of  this  ' '  Bacillus  icteroides, '  ' 
and  obtained  symptoms  which  he  believed  simulated  closely  those  of 
yellow  fever.  The  claims  of  Sanarelli  at  first  found  much  apparent 
confirmation,  but  later  work  by  Durham  and  Myers,13  Otto,1* 
Agramonte,15  and  others  has  definitely  refuted  his  original  claims, 
and  there  is  to-day  no  scientific  basis  for  the  assumption  that  the 
Bacillus  icteroides  has  any  etiological  relationship  to  the  disease 
Protozoan  incitants,  also,  have  been  described  by  Klebs,16  Schiiller,17 
Thayer,18  and  others,  without  bringing  conviction  or  even  justifying 
extensive  investigation  of  their  claims. 

While  the  earlier  etiological  investigations,  therefore,  were  in- 
conclusive, much  evidence  was  adduced  which  seemed  to  indicate 
that  the  virus  was  filtrable.  Reed,  Carroll,  Agramonte  and  Lazear, 
carried  out  experiments  which  they  thought  demonstrated  that  the 
infecting  agent  was  present  in  the  blood  of  patients  during  the 
first  three  days  of  the  disease,  and  could  pass  through  the  pores  of 


10  Cornil  and  Babes,  Comptes  rend,  de  1'acad.  des  sci.,  1883. 
"Sternberg,  Cent.  f.  Bakt.,  I,  xii,  1897. 

12  Sanarelli,  Ann.  de  1'inst.   Pasteur,   1897,   and  Cent.  f.  Bakt.,  I,  xxii,  xxvii, 
and  xxix. 

"Durham  and  Myers,  Thompson  Yates  Laboratory  Eeports,  3,  1902. 

14  Otto,  Vierteljahrsch.  f .  gericht.  Medizin,  etc.,  27,  1904. 

15  Agramonte,  N.  Y.  Med.  News,  1900. 
"Klebs,  Jour.  Am.  Med.  Assn.,  April,  1898. 
"Schiiller,  Berl.  klin.  Woch.,  7,  1906. 

18  Thayer,  Med.  Record,  1907. 


YELLOW  FEVER  AND  THE  LEPTOSPIRA  881 

a  Berkefeld  filter.  The  filtrability  of  the  virus  has  recently  become 
doubtful  in  view  of  the  researches  of  Noguchi. 

Eecently,  Noguchi19  has  carried  out  investigations  which  promise 
to  settle  the  etiological  problem  in  yellow  fever  conclusively. 
Noguchi  in  1918  carried  out  extensive  studies  in  the  Yellow 
Fever  Hospital  at  Guayaquil,  where  he  began  by  observing  172 
typical  cases  of  the  disease,  studying  them  clinically  and  patholog- 
ically. We  mention  this  because  the  only  possible  source  of  error 
in  his  investigations  seems  to  us  to  be  that  of  mistaking  of  cases 
of  infectious  jaundice  for  yellow  fever,  which,  of  course,  is  possible 
in  view  of  the  clinical  similarity  between  the  diseases,  as  emphasized 
by  Nishi.  It  is  important,  therefore,  to  mention  that  Noguchi 
worked  on  what  he  considered  classical  cases  of  yellow  fever  in  a 
Yellow  Fever  Hospital  where  he  was  aided  by  physicians  familiar 
with  the  disease.  He  began  by  injecting  blood  from  these  cases 
in  the  first  week  of  the  disease,  into  a  large  number  of  different 
animals.  The  only  animals  with  which  he  had  success,  however, 
were  guinea-pigs.  With  the  blood  of  early  cases  he  inoculated 
seventy-four  guinea-pigs  from  twenty-seven  cases  of  yellow  fever. 
Of  these,  eight,  representing  six  cases,  came  down  with  symptoms 
resembling  human  yellow  fever.  After  an  incubation  period  of  three 
to  six  days,  the  guinea-pigs  showed  a  marked  rise  of  temperature, 
eonjunctival  congestion,  leucocytosis  followed  by  progressive  leuco- 
penia,  and  a  drop  of  temperature  after  a  few  days.  Jaundice  was 
noticed  during  this  period,  and  hemorrhages  from  the  nose  and  anus 
were  occasionally  observed.  At  autopsy  the  tissues  were  deeply 
jaundiced  and  the  organs  hyperemic.  Hemorrhagic  spots  were 
found  in  the  lungs  and  in  the  intestinal  mucous  membrane. 

In  the  blood,  liver  and  kidneys  of  the  guinea-pigs  experimentally 
infected,  Noguchi  found  the  organism  which  he  calls  the  Leptospira 
Ictero-Hcemorrhagice.  This  organism  resembles  quite  closely  the 
causative  agent  of  infectious  jaundice.  In  general,  his  monkey  ex- 
periments were  negative  in  all  ,species  except  the  Marmosets  (Midas 
Oedipus  and  Midas  Geoff royi). 

The  examination  of  the  blood  of  patients  by  the  dark  field  in 
Noguchi 's20  hands  never  yielded  large  numbers  of  organisms.  In 
careful  examinations  made  on  twenty-seven  cases  he  found  them 
in  three  only.  He  never  saw  them  in  urine,  but  one  guinea-pig 

19  Noguchi,  Jour.  Exper.  Med.,  29,  1919,  547-596. 

20  Noguchi,  Jour.  Exper.  Med.,  30,   1919,  87. 


882  PATHOGENIC   MICROORGANISMS 

inoculated  with  10  c.c.  of  urine  came  down.  Examinations  of  the 
organs  revealed  them  in  only  one  case  in  the  kidneys. 

Working  with  mosquitoes,  Noguchi  allowed  Stegomyia  calopus 
to  bite  yellow  fever  patients  during  the  early  stages  of  the  disease. 
He  placed  the  arm  of  a  patient  into  a  cage  containing  200  to  300 
mosquitoes  hatched  from  the  larvae,  and  allowed  the  mosquitoes 
to  feed  until  the  females  were  full  of  blood.  Twenty-three  days 
after  feeding  on  the  patient,  the  mosquitoes  were  allowed  to  feed 
on  guinea-pigs.  By  this  method  he  claims  that  he  obtained  one 
positive  experiment  out  of  six.  In  this  positive  experiment  the 
guinea-pigs  developed  typical  symptoms  in  about  fifteen  days. 

Noguchi  also  cultivated  the  Leptospira  three  times  directly  from 
yellow  fever  patients.  The  medium  consisted  of  a  mixture  of  one 
part  of  serum  from  non-immune  persons  and  three  parts  of  Ringer 's 
solution,  used  both  in  the  liquid  form  and  also  in  the  semi-solid 
condition,  by  adding  small  amounts  of  melted  neutral  agar.  To 
this  about  1  c.c.  of  citrated  blood  from  the  median  basilic  vein  of 
the  patient  was  added,  first  being  mixed  with  the  semi-solid  agar, 
while  this  was  in  the  fluid  condition  at  42°  C.  This  was  allowed 
to  solidify  by  cooling,  and  the  warm  Ringer's  solution  was  then 
poured  on  the  semi-solid  portion  and  about  0.5  to  1  c.c.  of  the  same 
blood  introduced.  The  culture  was  then  covered  with  paraffin  oil. 

He  also  cultivated  the  organisms  from  infected  guinea-pigs. 

He  described  them  as  follows:  The  Leptospira  is  an  extremely 
delicate  filament  about  4  to  9  micra  in  length  and  0.2  of  a  micron 
in  width.  It  tapers  gradually  toward  the  extremities  and  ends 
in  thick  sharp  points.  It  is  minutely  wound  at  short  and  regular 
intervals,  each  section  measuring  about  0.25  of  a  micron.  The 
windings  are  so  placed  as  to  form  a  zigzag  line  at  angles  of  90°. 

It  is  not  visible  by  ordinary  light,  but  is  easily  seen  with  a  dark 
field.  It  is  actively  motile,  showing  a  vibratory  motion  and  some- 
times twisting  parts  of  the  filament.  It  bores  into  the  semi-solid 
material  and  is  remarkably  flexible. 

It  is  difficult  to  stain  with  ordinary  dyes,  but  can  be  fixed  by  osmic 
acid  and  stained  with  Giemsa  or  other  polychrome  stains. 

In  regard  to  transmission,  it  is  an  important  fact  (Noguchi) 
that  67  per  cent  of  the  wild  rats  of  Guayaquil  showed  organisms 
in  their  kidneys  similar  in  appearance  to  the  leptospira  just 
described;  and  these  inoculated  into  guinea-pigs  produced  lesions 
similar  to  those  produced  by  the  yellow  fever  blood. 


YELLOW  FEVER  AND  THE  LEPTOSPIRA  883 

In  regard  to  the  question  of  the  identity  of  the  yellow  fever 
spirochaete  with  that  of  Weil's  disease,  Noguchi  states  that  serolog- 
ical  differentiation  could  be  made.  Polyvalent  immune  sera,  one 
specific  for  icteroides  and  the  other  specific  for  icterohemorrhagice, 
showed  a  high  neutralizing  power  for  cultures  of  the  homologous 
group.  He  found,  however,  that  the  action  of  the  sera  is  not 
absolutely  specific,  since  the  injection  of  a  sufficient  amount  of 
anti-icteroides  serum  prevents  a  fatal  outcome  in  guinea-pigs  in- 
oculated with  multiple  doses  of  the  other  organism,  and  vice  versa. 
Other  forms  of  serum  reaction  showed  the  same  lack  of  absolute 
specificity. 

Subsequently,  Noguchi  attempted  to  protect  guinea-pigs  against 
multiple  doses  by  injection  of  immune  sera.  He  produced  polyvalent 
immune  serum  by  inoculation  of  a  horse,  and  found  that  such  serum 
could  protect  guinea-pigs  when  administered  during  the  period  of 
incubation,  and  modified  the  course  of  the  disease  when  used  in 
the  early  stages,  but  had  no  perceptible  result  in  the  later  periods. 

Subsequent  to  the  experiments  of  Noguchi  recorded  above, 
Noguchi  and  Kligler21  obtained  results  of  similar  experimental  sig- 
nificance in  Yucatan. 

PREVENTION  OF  YELLOW  FEVER 

Success  in  the  prevention  of  yellow  fever  has  been  one  of  the 
important  factors  in  the  development  of  South  and  Central  Ameri- 
can countries.  The  conversion  of  Rio  de  Janeiro  into  a  healthy 
port  by  Oswaldo  Cruz,  the  cleaning  up  of  New  Orleans  and  the 
work  done  by  Gorgas  in  Panama  indicate  the  splendid  role  which 
an  understanding  of  the  relations  of  transmission  in  this  disease 
have  played  in  the  progress  of  civilization.  Knowing  what  we  do 
about  the  Stegomyia  and  the  part  played  by  it  in  transmission, 
preventive  measures  must  depend  primarily  upon  the  suppression 
of  the  mosquito  and  the  isolation  of  cases  from  which  mosquitoes 
may  acquire  the  infection.  The  disease  is  never,  as  far  as  we  know, 
conveyed  by  direct  infection  from  man  to  man,  or  by  soiled  clothing, 
etc.,  and  in  consequence  there  is  no  necessity  for  care  in  these 
matters.  The  stegmoyia  breeds  particularly  in  rather  pure  water, 
such  as  found  in  rain  water  cisterns,  and  fortunately  does  not,  like 
some  other  mosquitoes,  breed  in  swamps,  ponds  and  other  natural 


NogucM  and  Kligler,  Jour.  Exper.  Med.,  32,  1920,  601. 


884  PATHOGENIC   MICROORGANISMS 

surface  waters.  Its  flying  radius  is  apparently  not  very  large. 
Unlike  the  Anopheles,  its  habits  are  diurnal,  instead  of  nocturnal. 
Rosenau  states  that  experience  of  the  epidemic  in  New  Orleans  in 
1905  showed  that  this  mosquito  docs  not  fly  far  from  its  place  of 
birth.  We  take  from  the  same  writer  the  statement  that,  in  order 
to  hold  back  the  small  mosquito,  a  mesh  must  be  used  containing 
at  least  twenty  strands  to  the  inch. 

Prevention  may,  therefore,  be  summed  up  as  consisting  in  screen- 
ing of  patients,  screening  of  houses,  destruction  of  mosquitoes  within 
houses  by  insecticides  and  fumigation  and  the  painstaking  removal 
of  all  stagnant  waters  in  the  neighborhood  of  human  habitations 
with  especial  care  to  the  screening  of  drinking  water  cisterns  in 
places  where,  as  in  Bermuda,  drinking  water  is  collected  in  rain 
water  receptacles.  In  places  like  Bermuda,  the  Stegomyia  fasciata 
is  common,  and  yet  there  has  been  no  case  of  yellow  fever,  we 
understand,  since  1869. 

Immunity. — Natural  immunity  against  yellow  fever  was  formerly 
assumed  to  exist  in  the  negro  race.  More  recent  investigations  have 
not  borne  out  this  assumption.  The  negro  soldiers  of  the  American 
army  in  Cuba  were  afflicted  equally  with  the  white  troops.  The 
relative  immunity  of  dark-skinned  races,  however,  is  explained  pos- 
sibly by  the  fact  that  the  stegomyia  prefers  to  attack  light-colored 
surfaces. 

A  single  attack  seems  to  protect  against  subsequent  infection 
throughout  life. 

Relative  immunity  was  produced  by  Marchoux,  Salimbeni,  and 
Simond,22  by  injections  of  the  serum  of  convalescents,  serum  heated 
to  55°  C.,  and  of  defibrinated  blood  preserved  for  eight  days  in 
vessels  sealed  with  vaseline. 

Experimental  work  is  being  carried  out  by  Noguchi  on  both 
active  and  passive  immunization  by  the  use  of  his  pure  cultures 
of  the  Leptospira  icteroides.  These  experiments  are  being  actively 
carried  out  at  the  present  time,  but  final  results  have  not  yet  been 
achieved.  On  the  whole,  however,  the  promise  of  this  work  is  great 
and  extremely  encouraging.  It  is  especially  encouraging  in  con- 
sideration of  the  great  similarity  between  this  Leptospira  and  the 
one  of  Weil's  disease  with  which  immunization  experiments  are  also 
full  of  promise. 

22  Marchoux,  Salimbeni,  and  Simond,  Ann.  <le  1'mst.  Pnsteur,  -190.3. 


YELLOW  FEVER  AND  THE  LEPTOSPIRA  885 


WEIL'S    DISEASE;    ICTERO-KflSMORRHAGIC    FEVER; 
(EPIDEMIC    JAUNDICE) 

This  is  a  disease  which  has  been  known  for  a  very  long  time. 
It  was  described  by  Larrey  as  early  as  1800  and  the  probability 
of  its  being  a  clinical  entity  was  recognized  by  a  number  of  other 
observers  between  that  time  and  1886  when  Weil  gave  an  accurate 
description  of  a  number  of  cases  he  observed  in  Germany.  The 
infectious  nature  of  the  disease  was  suspected  from  the  course  and 
symptoms  and  many  etiological  "guesses"  were  made.  For  a  time 
it  was  suspected  that  the  disease  was  particularly  associated  with 
butcher  shops  and  occurred  especially  in  individuals  following 
the  butcher's  trade.  During  the  recent  war  the  disease  occurred 
in  many  of  the  armies  at  the  front,  in  the  trenches.  They  were 
first  reported  from  the  Italian  front  and  later  in  the  British,  German 
and  French  armies,  occurring  at  times  in  almost  epidemic-like  form. 
The  disease  has  been  relatively  mild  in  European  countries,  whereas 
in  Japan  where  it  seems  to  be  particularly  a  disease  of  mine  workers, 
it  is  quite  virulent  and  often  has  a  high  death  rate.  It  seems  to 
have  been  particularly  a  disease  of  war,  since  the  cases  of  Larrey 
came  from  the  Eygptian  army  of  Napoleon.  The  recent  outbreaks 
were  largely  among  soldiers  and  it  is  stated  that  a  considerable 
number  of  cases  occurred  in  the  South  African  and  the  American 
Civil  Wars.  It  is  important  to  note  in  connection  with  what  will 
be  said  later  about  yellow  fever  that  Nishi  identified  Weil's  disease 
with  fellow  fever  some  time  ago,  but  was  opposed  by  Ohno.  Inada 
and  his  co-workers  mention  this,  and  emphasize  the  difference  be- 
tween the  two  diseases  from  the  epidemiological  point  of  view, 
though  they  admit  the  similarity  of  the  symptoms.  Also,  transmis- 
sion seems  to  be  quite  different  in  the  two  diseases. 

The  Disease. — Fiedler,23  whom  we  quote  from  Inada24  divides 
the  disease  into  three  stages,  an  initial  period  of  two  or  three  days, 
with  fever,  followed  by  a  second  stage  of  three  days,  during  which 
icterus,  edema  of  the  liver,  enlargement  of  the  spleen  and  skin 
hemorrhages  occur.  The  defervescent  period  starts  on  the  seventh 
day  and  initiates  convalescence.  Inada  describes  it  as  follows:  The 


"Fiedler,  Deut.  arch.  klin.  Med.,  1892,  1,  232. 
"Inada,  Jour,  exper.  Med.,  26,  1917,  355. 


886  PATHOGENIC   MICROORGANISMS 

onset  is  initiated  by  chills  and  fever,  intestinal  disturbances,  head- 
ache, muscular  pains,  hyperemia  of  the  conjunctivas  and  al- 
buminuria.  In  this  stage  death  is  rare,  and  during  this  time  the 
spirocha3tes  are  freely  circulating  in  the  blood.  Blood  injected 
during  this  period  into  guinea-pigs  produces  a  typical  reaction. 
The  infectivity  of  the  blood  decreases  from  this  time  on,  according 
to  Inada.  From  the  seventh  to  the  thirteenth  day  of  the  icteric 
period,  which,  according  to  Inada,  covers  a  little  less  than  a  week. 
The  symptoms  decrease,  the  jaundice  and  hemorrhages  into  the 
skin  appear,  together  with  great  weakness,  nervous  and  cardiac 
symptoms.  The  two  stages  shade  into  each  other  and  the  second 
period  is  the  one  during  which  death  is  most  common.  In  cases 
dead  at  this  time  the  spirochaetes  have  disappeared  from  the  blood, 
and  antibodies  can  be  shown  by  the  Pfeiffer  test.  During  the  second 
stage,  the  spirochaetes  are  present  in  the  urine  and  can  be  found 
by  the  dark  field.  Inada  showed  them  in  17.4  per  cent  of  his  cases 
on  the  tenth  day,  with  a  gradually  growing  percentage  up  to  52.2 
per  cent.  As  antibodies  develop  the  spirochaetes  disappear  from 
the  blood  and  from  the  liver. 

On  the  thirteenth  and  fourteenth  day  begins  the  convalescent 
stage.  The  jaundice  becomes  less  and  anemia  and  emaciation 
appear.  Antibodies  reach  their  highest  point  in  the  blood,  the 
spirochaetes  completely  disappear  from  the  blood,  and  become  more 
abundant  in  the  urine.  Inada  states  that  the  only  organ  in  which 
the  spirochaetes  can  be  found  at  this  time  are  the  kidneys.  By 
the  nineteenth  and  twentieth  day,  practically  all  cases  show  the 
organisms  in  the  urine. 

The  incubation  period  of  the  disease  is  five  to  seven  days. 

Etiology  of  Weil's  Disease. — A  great  many  different  microorgan- 
isms have  been  described  in  the  course  of  time  as  the  cause  of 
Weil's  disease.  The  question  was  definitely  settled  in  1916  by  Inada, 
Yutaka,  Hoki,  Kaneko  and  Ito25  who  described  the  Spirochoeta  Ictero- 
Haemorrhagiae.  They  found  the  organisms  in  the  blood,  liver,  adrenal 
glands  and  kidneys  and  transmitted  it  by  intraperitoneal  injection 
into  guinea-pigs.  They  also  succeeded  in  cultivating  the  organisms 
by  the  Noguchi  method.  There  can  be  no  question  whatever  at 
the  present  time  concerning  the  etiologieal  importance  of  this  or- 
ganism in  Weil's  disease.  Tnadn  ;md  his  en-workers  inoculated 


K  Inada,  and  coworkers,  Jour.  Exper.  Med.,  23,  1916,  377. 


YELLOW  FEVER  AND  THE  LEPTOSPIRA  887 

monkeys,  rabbits,  rats  and  guinea-pigs  with  the  blood  of  patients 
in  July,  1913,  and  found  that  the  guinea-pigs  developed  albuminuria, 
Jaundice  and  hemorrhages  when  the  blood  was  injected  during  the 
first  seven  days  of  the  illness.  Subsequently,  they  found  the  spiro- 
chaetes  in  large  numbers  in  the  livers  of  inoculated  guinea-pigs, 
and  following  this,  found  them  microscopically  in  six  specimens  of 
patients'  blood  and  at  autopsies.  The  jaundice  in  the  guinea-pigs 
appears  about  seven  or  eight  days  after  inoculation,  the  shortest 
time  being  six  days  and  the  longest  thirteen.  The  disease  can  be 
kept  going  in  guinea-pigs  through  many  generations.  Rabbits  are 
insusceptible.  Mice  and  rats  seem  to  be  slightly  susceptible. 

The  organism  seems  always  to  remain  outside  the  blood  cells 
and  is  present  in  the  interstitial  tissues  of  organs.  It  has  irregular 
wavy  curves  and  is  from  6  to  9  micra  long,  the  largest  being  12 
to  13.  Thickness  seems  to  vary  with  staining  methods.  Undula- 
tions are  more  irregular  than  those  of  treponema  pallidum  and  are 
usually  composed  of  two  or  three  large  irregular  and  four  or  five 
smaller  waves. 

Cultivation  was  successful  in  the  hands  of  Inada  and  his  co- 
workers  by  the  method  used  by  Noguchi  in  the  case  of  the  spiro- 
chaete  of  recurrent  fever.  They  used  guinea-pig  instead  of  rabbit 
kidney,  and  covered  their  deep  tubes  with  liquid  paraffin.  They 
found,  however,  that  37°  C.  is  not  the  best  temperature  for  develop- 
ment, but  that  the  cultures  grow  best  at  between  22°  and  25°  C. 
The  cultures  are  odorless  and  there  is  no  coagulation  of  the 
fluid  which  remains  clear,  even  slight  cloudiness  indicating  con- 
tamination. The  cultures  may  live  from  three  to  six  weeks  in  the 
first  generation,  and  in  later  generations  as  long  as  fifty-five  days. 

Transmission. — Subsequent  to  the  announcement  of  Inada 's  dis- 
covery, Miyjajima  called  attention  to  the  fact  that  he  had  seen 
spirochaetes  in  the  kidneys  of  field  mice.  This  clue  was  followed 
up  by  Ido,  Hoki,  Ito,  Wani26  and  many  others  who  found  spirochaetes 
similar  to  the  ones  described  by  Inada  in  house  and  roof  rats,  Epymis  or 
Mus  alexandrinus  and  Mus  norvegicus.  Since  that  time  Stokes27  demon- 
strated the  organisms  in  the  kidneys  of  field  rats  in  the  neighborhood 
of  infected  districts  in  Flanders,  and  Noguchi28  has  found  similar 


24  Ido,  Hoki,  Ito,  and  Wani,  Jour.  Exper.  Med.,  26,  1917,  341. 
27  Stokes,  Eyle  and  Tytler,  Lancet,  1,  1917,  142. 
2SNo(/uchi,  Jour.  Exper.  Med.,  25,  1917,  755. 


888  PATHOGENIC   MICROORGANISMS 

organisms  in  wild  rats  in  the  United  States.  From  these  and  other 
experiments,  it  appears  probable  that  the  disease  is  primarily  one 
of  rats,  and  is  transmitted  secondarily  to  man.  It  may  be  trans- 
mitted by  contact  with  the  urine  of  the  rats  and,  according  to  the 
Japanese  investigators,  the  infected  soil  may  play  an  important  part. 
Damp,  cold  mines  in  which  rats  abound  seem  to  be  particularly 
favorable.  Infection  from  man  to  man  may  occur,  since,  as  we  have 
seen,  the  spirochaetes  pass  into  the  urine,  but  this  is  relatively  rare. 
According  to  Inada  and  his  coworkers,29  the  spirochaetes  enter  the- 
human  body  chiefly  through  the  alimentary  canal,  but  they  may  also 
come  in  through  abrasions  of  the  skin.  Among  fifty-five  cases  ad- 
mitted to  Inada 's  clinic,  only  a  few  indicated  cutaneous  origin,  but 
there  were  certain  of  these  in  which  cutaneous  origin  could  not 
be  denied. 

Serum  Treatment. — Inada  and  Ido29  first  attempted  to  treat  the 
disease  with  the  blood  of  convalescent  cases  and  observed  astonish- 
ingly favorable  results.  Later,  they  actively  immunized  horses  with 
pure  cultures  and  finally  obtained  horses  into  whom  they  could 
inject  800  c.c.  of  a  pure  culture  containing  thirty  spirochaetes  per 
field.  0.01  c.c.  of  this  serum  injected  into  the  peritoneal  cavity  of 
guinea-pigs  protected  them  against  1  c.c.  of  pure  culture.  In  human 
cases  they  injected  as  much  as  60  c.c.  of  this  horse  serum  in  twenty- 
four  hours.  Of  thirty-five  patients  who  received  serum,  seven  died, 
showing  a  mortality  of  11.4  per  cent  as  against  17.3  per  cent  of 
cases  dead  at  the  same  time,  without  serum.  The  serum  completely 
destroyed  spirochaetes  contained  in  the  circulating  blood.  Further 
work  on  specific  treatment  is  in  progress. 

Prevention. — The  prevention  of  the  fever  seems  from  the  above 
facts  to  rest  primarily  upon  the  destruction  of  rats  and  the  preven- 
tion of  contact  of  rats  with  food,  the  prevention  of  the  pollution  of 
soil,  and  isolation  and  care  in  regard  to  transmission  in  the  case  of 
patients  suffering  from  the  disease. 

"Inada  and  Ido,  Jour.  Exper.  Med.,  24,  1916,  465. 


SECTION    IV 

DISEASES  CAUSED  BY  FILTRABLE  VIRUS,  THE  EXANTHE- 
MATA, AND  DISEASES  OF  UNCERTAIN  ETIOLOGY 


CHAPTER    XLV 

GENERAL   CONSIDERATION   OF   FILTRABLE   VIRUS,    SMALLPOX   AND 

RABIES 

FILTRABLE  VIRUS 

RECENT  investigations  into  the  causation  of  disease  have  revealed 
that  a  considerable  number  of  infections  may  be  caused  by  organisms 
too  small  to  be  held  back  by  niters  through  which  even  the  smallest 
bacteria  cannot  pass.  The  earliest  observations  of  such  "filtrable 
virus"  are  probably  those  of  Frosch  (1898)  in  foot-and-mouth  dis- 
ease and  of  Beijerinck  in  the  mosaic  disease  of  tobacco.  Since  then 
similar  investigations  have  shown  that  a  large  number  of  diseases 
are  probably  caused  by  such  minute  organisms;  their  investigation, 
loDg  delayed  by  the  belief  in  their  invisibility  by  even  the  most 
powerful  microscopic  aid,  and  by  our  inability  to  cultivate  them, 
has  taken  new  impetus  from  the  discovery  and  probable  cultivation 
of  minute  globoid  bodies  from  the  virus  of  poliomyelitis  by  Flexner 
and  Noguchi  (see  below). 

Similar  bodies  have  since  then  been  seen  in  connection  with 
other  diseases  by  many  observers,  and  in  lethargic  encephalitis 
recently  Strauss  and  Loewe  claim  to  have  cultivated  organisms  quite 
similar  to  those  of  poliomyelitis  cultivated  by  Noguchi.  In  thinking 
of  filtrability,  however,  one  must  remember  that  filtrability,  like 
diffusibility,  is  not  an  absolute  concept,  and  that  much  depends 
upon  the  nature  of  the  filter  used  and  the  amount  of  suction  applied. 
Thus,  there  has  been  much  discussion  as  to  the  filtrability  of  diseases 
like  syphilis,  yellow  fever,  etc.,  and  in  conditions  caused  by  flexible 

889 


890  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

organisms  like  spirochaetes  it  might  be  quite  possible  with  sufficient 
suction  and  niters  of  sufficient  size  to  mechanically  pull  through 
organisms  which,  by  their  actual  normal  dimensions,  would  be  re- 
garded as  too  large  to  be  classified  with  filtrable  organisms  merely 
in  regard  to  size.  The  Berkefeld  filters  of  va-rious  grades  will  differ 
materially  in  the  sizes  of  the  particles  they  will  let  through,  and 
filters  that  are  new  will  act  differently  from  filters  that  have  been 
used;  and  in  this  regard  will  vary  to  some  extent,  according  to 
the  nature  of  the  substances  that  they  have  been  used  for  at  previous 
filtrations.  Also,  in  a  comparative  experiment  made  by  us  in  con- 
nection with  syphilis  some  years  ago,  it  was  found  that  new  filters 
of  presumably  the  same  manufacturer's  grade,  differed  somewhat 
in  regard  to  the  substances  they  allowed  to  pass  through,  and  all 
experienced  investigators  will  always  control  their  filtration  experi- 
ments by  adding  to  the  substance,  the  filtrability  of  which  they  wish 
to  test,  bacteria  of  a  small  size  (like  influenza  bacillus  or  B.  pyo- 
cyaneus),  so  that  they  may  know  in  that  particular  filtration  the 
filter  used  was  impermeable  to  the  small  bacteria.  Filtrability,  then, 
is  a  matter  of  gradation  and  not  a  sharp  wall  which  necessarily 
separates  one  kingdom  of  microorganisms  from  another. 

The  following  tabulation  of  the  diseases  presumably  caused  by 
filtrable  virus  was  made  by  Wolbach1  some  time  ago  and  is  taken 
from  his  article  by  us  in  its  entirety  with  one  or  two  modifications 
made  necessary  by  recent  developments: 


SMALLPOX 

Smallpox  or  variola  is  one  of  the  most  virulent  of  infectious 
diseases.  Throughout  history  it  has  been  a  severe  scourge  of  man- 
kind, prevailing  in  China  and  other  Eastern  countries  many  centuries 
before  Christ  and  sweeping  through  medieval  Europe,  especially  at 
the  time  of  the  Crusades,  in  a  series  of  severe  epidemics.  All  races 
of  men  are  susceptible  and  no  age  from  childhood  to  senility  is 
exempt.  In  modern  times  the  disease  is  endemic  in  most  uncivilized 
countries,  especially  those  of  the  East,  and  occurs  sporadically  in 
all  parts  of  the  globe.  Owing  to  rigid  enforcement  of  vaccination 
and  of  quarantine  laws,  however,  the  disease  has  been  practically 
eradicated  from  civilized  countries. 


1  Wolbach,  Jour,  of  Med.  Ees.,  xxvii,  1912. 


GENERAL   CONSIDERATION    OF   FILTRABLE   VIRUS 


891 


Disease  * 

TRANSMISSION 

Occurrence 

Direct 

Indirect 

Molluscum  contagiosum.  .  . 

Direct  contact 

Man 

Culex  fatigans 

Man 

Verruca  vulgaris  filtrability? 

? 

Man 

Trachoma  filtrability?  

Direct 

Man 

Poliomyelitis  

Unknown;    probably 
nasal,  etc.,  discharges 

Indirect  by  stable-fly 

Man 

Measles  filtrability  claimed 
by  Goldberger  and  Ander- 

Direct 

Man 

Scarlet     fever?      filtrability 
claimed  by  Cantacuzene 
and     Bernhardt     but 
doubtful? 

Probably  direct 

Man 
Chimpanzee 

Foot-and-mouth  disease.  .  . 

Direct 

Man,  cattle  and  swine 

Rabies  

Direct    by    bite    with 
saliva 

Man    and    all    mam- 
mals; birds 

Variola  and  vaccinia  

Direct 

Man,     cattle;      trans- 
mitted  to   monkeys, 
rabbits 

Pleuro-pneumonia  of  cattle. 

Direct 

Bovine  species 

Probably  insects,  mos- 
quitoes 

Horses 

Sheep-pox  

Direct 

Sheep 

Food    contaminated 
with  excreta 

Cattle 

Hog-cholera  

Direct 

Hogs 

Swamp  fever  of  horses  

Probably    indirect    by 
insects 

Horses 

Agalactia  of  sheep  and  goats 

Contact 

Sheep  and  goats 

"  Blue  tongue  "  

? 

? 

Sheep 

Guinea-pig  epizootic  

? 

? 

Guinea-pigs 

Guinea-pig  paralysis  

? 

? 

Guinea-pigs 

Novy's  rat  disease  

? 

? 

Rats 

Fowl  pest  

Feces 

Pheasants,     sparrows, 
geese 

Fowl  diphtheria 

Contact  exudates,  etc  . 

Fowl 

Rous's  chicken  sarcoma...  . 

? 

? 

Chickens 

Pappatacai    (three-day 
fever) 

Unknown 

Phlebotomus  papatasii 

Man 

Influenza,    claimed   by   Ya- 
manouchi,     Olitsky     and 
others  

Direct 

Man 

Mumns,   claimed  by  Woll- 

stein  

Direct 

*  Table  slightly  modified  from  Wolbach,  loc.  cit. 


892  DISEASES  CAUSED   BY  FTLTRARLE  VIRUS 

The  etiological  factor  which  causes  smallpox  is  still  unknown. 
Numerous  researches  aimed  at  the  discovery  of  cultivatable  micro- 
organisms in  the  lesions  or  blood  of  infected  patients  have  met  with 
uniform  failure.  Streptococci,  though  often  found  in  the  smallpox 
vesicles  and  pustules,  and  often  undoubtedly  contributing  materially 
to  the  fatal  outcome  of  the  disease,  may  be  regarded  as  purely 
secondary  in  significance. 

Communications  which  have  claimed  the  discovery  of  a  protozoan 
incitant  of  the  disease  have,  on  the  other  hand,  been  numerous  and, 
in  some  cases,  have  seemed  plausible.  Yet  absolute  proof  has  always 
been  lacking.  The  literature  on  this  question  is  extensive  and  some 
of  the  earlier  contributions,  such  as  those  of  Griinhagen,2  of  Van 
der  Loeff,3  and  of  Pfeiffer,4  possess  historical  interest  only.  The 
work  which,  of  recent  years,  has  attracted  the  most  serious  attention 
to  this  subject  is  that  published  by  Guarnieri5  in  1892.  This  observer 
found,  in  the  deeper  cells  of  the  epithelium  covering  the  pustules, 
both  of  smallpox  lesions  and  of  vaccination  lesions,  small  bodies 
which  were  easily  stained  by  hematoxylin,  safranin,  or  carmin. 
Similar  bodies  could  be  observed  in  the  cells  of  corneal  lesions 
experimentally  produced  in  rabbits.  Guaranieri  claimed  that  he 
distinguished  both  cytoplasm  and  nucleus  in  these  bodies  and 
described  both  binary  division  and  reproduction  by  sporulation  as 
in  the  sporozoa.  He  named  the  supposed  protozoan  ' '  Cy torryctes 
variolae. "  At  about  the  same  time  Monti6  described  similar  bodies 
in  the  cells  of  the  Malpighian  layer  of  the  skin  covering  smallpox 
lesions  and,  a  few  years  later,  Clarke7  confirmed  the  researches  of 
Guarnieri.  Subsequently,  many  researches  were  carried  out  on  the 
same  subject  in  this  country,  the  most  notable  being  those  of  Coun- 
cilman,8 Magrath  and  Brinckerhoff,  and  of  Calkins.9  The  former 
authors  came  to  the  conclusion  that  the  bodies  seen  by  Guarnieri 
were  parasites,  and  the  latter  author  even  described  a  distinct  life- 
cycle  for  these  parasites  comparable  to  that  of  some  protozoa. 

2  Griinhagen,  Arch.  f.  Dermat.  u.  Syph.,  1892. 
1  Van  der  Loeff,  Monat.  f.  prakt.  Dermat.,  iv. 
*L.  Pfeiffer,  Zeit.  f.  Hyg.,  xxiii. 

5  Guarnieri,  Arch,  per  le  sc.  med.,  xxvi,  1892 ;  Cent,  f .  Bakt.,  I,  xvi,  1894. 

6  Monti,  Cent.  f.  Bakt.,  I,  xvi. 

7  Clarice,  Brit.  Med.  Jour.,  2,   1894. 

8  Councilman,  Magrath,  and  Brinclccrhoff,  Jonr.  Med.  Res.,  xi,  1904. 

9  Calkins,  Jour.  Med.  Res.,,  xi,  1904. 


GENERAL   CONSIDERATION    OF   FILTRABLE   VIRUS          893 

These  researches,  however,  are  by  no  means  convincing,  and 
Ewing,10  while  admitting  that  the  vaccine  bodies  are  probably 
specific  for  variola,  calls  attention  to  the  fact  that  specific 
cell-degenerations  or  inclusions  are  found  in  diphtheria,  measles, 
glanders,  rabies,  and  other  infectious  processes,  which  can  not  be 
regarded  as  in  any  way  related  to  these  diseases  etiologically,  and 
suggests  the  probability  of  a  similar  interpretation  for  the  vaccine 
bodies.  Much  has  been  said  on  both  sides  of  the  question  since 
that  time,  and  the  problem  can  not  be  regarded  as  settled.  The 
burden  of  proof,  of  course,  rests  upon  those  who  claim  the  discovery 
of  a  specific  microorganism,  and  absolute  proof  will  probably  be 
lacking  until  our  experimental  methods  are  such  as  will  permit  of 
other  than  purely  morphological  demonstration. 

Whatever  the  actual  causative  agent  may  be,  it  is  certain  that 
the  disease  is  transmitted  with  extreme  ease — actual  contact,  direct 
or  indirect,  with  a  patient  being  unnecessary  for  its  transmission. 
While  we  have  no  certain  knowledge  of  the  portal  of  entry  through 
which  the  virus  invades  the  human  body,  many  considerations  have 
made  it  seem  plausible  that  this  may  take  place  through  the  mucosa 
of  the  upper  respiratory  tract. 

Our  knowledge  of  the  means  of  defense  against  the  malady  is 
fortunately  more  advanced  than  is  that  of  its  etiology.  It  has  been 
known  for  centuries  that  one  attack  of  smallpox  protects  against 
subsequent  attacks.  This  knowledge  was  made  use  of  by  the  physi- 
cians of  ancient  China  and  India,  who,  during  mild  epidemics, 
exposed  healthy  children  to  infection,  hoping  that  mild  attacks 
would  result  which  would  confer  immunity.  While  dangerous  in 
the  extreme,  such  "variolation,"  nevertheless,  was  not  without  some 
benefit  and  was  even  introduced  into  Europe  in  the  eighteenth  cen- 
tury by  Lady  Mary  Wortley  Montagu. 

Such  practices,  however,  were  made  unnecessary  by  the  classical 
investigations  of  Jenner  published  in  1798.  Jenner,  as  a  student, 
had  been  impressed  with  the  fact  that  country-people  who  had  been 
infected  with  a  disease  known  as  cowpox,  were  usually  immune 
against  smallpox.  His  studies  and  observations  came  to  a  practical 
issue  when,  in  1796,  he  inoculated  a  boy,  James  Phipps,  with  pus 
from  a  cowpox  lesion  on  the  hand  of  an  infected  dairy-maid.  Two 
months  later  the  same  boy  was  inoculated  with  material  from  a 


10  Ewing,  Jour.  Mecl.  Res.,  xiii,  1905. 


894  DISEASES  CAUSED  BY  F1LTRABLE  VIRUS 

smallpox  pustule  without  subsequent  disease.  With  this  experiment 
the  principles  of  vaccination  as  in  use  at  the  present  time  were 
founded. 

The  question  as  to  the  identity  of  cowpox  and  smallpox  has  been 
the  basis  of  a  long  controversy.  Many  observers  claimed  from  the 
beginning  that  the  two  diseases,  though  closely  related  to  each  other, 
were  essentially  different.  Others,  on  the  contrary,  and  this  seems 
to  be  the  prevailing  opinion  among  scientists  at  the  present  dayv 
maintain  that  cowpox  or  vaccinia,  as  it  is  called  when  inoculated 
into  a  human  being,  represents  merely  an  altered  and  attenuated 
variety  of  variola.  This  latter  view  is  based  on  the  following  con- 
siderations, which  we  take  from  Haccius  as  quoted  by  Paul.11 

1.  Variola  is  invariably  transmissible  to  cattle,  when  proper  methods  of 
inoculation  are  employed. 

2.  Variola  carried  through  several   animals,  in  the  above  way,  becomes 
altered  in  character,  approaching  in  nature  typical  vaccinia  or  cowpox. 

3.  Such  virus,  reinoculated  into  man,  gives  rise  to  purely  local  lesions 
which  are  mild  and  unlike  smallpox. 

4.  Inoculation  with  such  virus  protects  both  man   and  animals  against 
subsequent  inoculation  with  cowpox,  and,  in  the  case  of  man,  against  smallpox 
as  well. 

Kolmer12  has  carried  out  complement-fixations,  using  as  antigens 
salt  solution  suspensions  of  cowpox  and  smallpox  virus,  and  has 
demonstrated  close  biological  relationship  between  the  two. 

It  has  been  claimed,  moreover,  that  cowpox  originally  was  trans- 
mitted to  cattle  by  human  beings  affected  with  smallpox.  This  seems 
likely  both  because  of  the  comparative  rarity  of  the  former  disease 
and  because  of  its  spontaneous  occurrence  almost  invariably  upon 
the  teats  of  cows,  although  both  males  and  females  are  equally 
susceptible  to  experimental  inoculation. 

The  relationship  of  variola  to  chicken-pox  or  varicella  has  been 
more  easily  determined.  Chicken-pox  does  not  protect  against  small- 
pox nor  is  this  the  case  vice  versa.  The  two  diseases  are  unquestion- 
ably quite  distinct. 

The  Production  of  Vaccine. — During  the  early  days  of  vaccina- 
tion, it  was  customary  to  inoculate  human  beings  with  the  matter 


11  Paul,  "Vaccination";  Kraus  and  Levaditi,  "Handbuch,    '  etc.,  L 
*- Kolmer,  Jour,  of  Immunology,  No.  1.  Feb.,  1916. 


GENERAL   CONSIDERATION   OF   FILTRABLE   VIRUS  895 

obtained  from  the  pustules  of  those  previously  vaccinated.  While 
this  method  was  perfectly  satisfactory  for  the  immediate  purposes 
in  view,  practical  difficulties  and  the  occasional  accidental  trans- 
mission of  syphilis  have  rendered  this  practice  undesirable.  In 
consequence,  at  all  institutes  at  which  vaccine  is  produced  for  use 
upon  man,  the  virus  is  obtained  from  animals.  Horses  and  mules, 
both  extremely  susceptible  to  vaccine,  have  been  employed,  and 
goats  have,  at  times,  been  chosen  because  of  their  insusceptibility 
to  tuberculosis.  Rabbits  have  also  been  used  more  recently  by  Cal- 
mette  and  Guerin.13 

The  animals  almost  exclusively  employed  at  the  present  day, 
however,  are  calves,  preferably  at  ages  of  from  six  months  to  two 
years.  Very  young  suckling  calves  are  unsuitable  because  of  the 
great  speed  of  development  and  small  size  of  the  lesions  produced.- 
The  animals  should  be  healthy  and  at  some  institutes  (Dresden) 
are  subjected,  before  use,  to  the  tuberculin  test ;  although,  according 
to  Paul,11  this  produces  a  hypersusceptibility  to  the  vaccine,  and 
can  be  omitted  without  danger  when  careful  supervision  is  observed. 
Some  observers  prefer  to  use  light-colored  animals  rather  than  dark- 
skinned  or  black  ones,  both  for  reasons  of  greater  ease  of  cleanliness 
and  because  the  former  are  supposed  to  be  more  susceptible  than 
the  latter.  This  contention  is  denied  by  others.  The  sex  of  the 
animals  is  immaterial. 

During  the  period  of  use,  the  calves  are  fed,  according  to  age, 
with  either  an  exclusive  milk  diet,  or  they  are  given,  in  addition, 
fresh  hay.  The  greatest  cleanliness  in  regard  to  the  bedding  and 
stalls  must  be  observed  and  separate  stables  should  be  available 
for  the  animals  under  treatment  and  those  under  observation  before 
treatment.  These  stables,  if  possible,  should  be  so  built  that  they 
can  be  easily  scoured  and  flushed  with  water,  and  stalls  should  be 
disinfected  after  occupation.  If  possible,  stables  should  be  artifi- 
cially heated  and  a  comfortable  temperature  maintained.  Halters 
and  fastenings  should  be  so  arranged  that  the  animals  can  not  lick 
the  scarified  surfaces.  Careful  veterinary  control  before  vaccination 
and  during  the  period  of  treatment  must  be  observed  in  order  to 
eliminate  animals  with  systemic  disease  or  other  complications. 

The  calves  may  be  vaccinated  with  material  taken  from 
previously  vaccinated  animals.  Again,  they  may  be  inoculated  with 


™Calmette  and  Guerin,  Ann.  de  1'inst.  Pasteur,  1902. 


896  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

"seed  virus"  obtained  from  the  vesicles  of  human  vaccinia.  This 
method  of  using  humanized  virus  for  the  inoculation  of  calves  for 
vaccine  production  is  preferred  by  many  workers  and  is  spoken  of 
as  "retrovaccination. " 

Park14  believes  that  the  most  efficient  and  reliable  seed  virus  con- 
sists of  what  he  calls  human-calf -rabbit  seed  virus.  Crusts  from 
healthy  children,  nineteen  days  after  vaccination,  are  collected. 
These  are  cut  up  and  emulsified  with  boiled  water.  With  this  an 
area  of  about  six  inches  square  is  inoculated  on  a  calf,  the  remainder 
of  which  is  vaccinated  the  ordinary  way.  The  virus  from  this 
space  is  separately  collected  and,  after  being  glycerinated,  is  used 
in  dilution  of  1  to  12i/£>  parts  of  salt  solution  to  vaccinate  rabbits 
on  the  shaven  skin  of  the  back.  The  pulp  from  this  rabbit  vaccina- 
tion is  then  used  for  calf  vaccination. 

Actual  vaccination  of  the  animals  is  done  as  follows:  Calves 
which  have  been  kept  under  observation  for  at  least  a  week  are 
thoroughly  washed  and  cleaned  and  the  abdomen  is  clipped  and 
shaved  over  an  area  extending  from  the  ensiform  cartilage  to  the 
pubic  region,  including  the  entire  width  of  the  belly  and  the  inner 
folds  of  the  thighs.  It  is  best  to  shave  the  animal  a  day  or  two 
before  vaccination  so  as  to  avoid  fresh  scratches  and  excoriations. 
Just  before  actual  operation  the  animal  is  strapped  to  a  specially 
constructed  operating  table  in  such  a  way  as  to  allow  free  access 
to  the  shaved  area.  This  area  is  now  thoroughly  washed  with  soap 
and  water  followed  by  alcohol,  or,  in  some  institutes,  by  a  weak 
solution  of  lysol.  If  the  latter  is  used,  the  field  of  operation  must 
again  be  thoroughly  rinsed  with  sterile  .water.  About  a  hundred 
small  scarifications  are  made  in  this  area,  preferably  by  crossed 
scratches,  covering  for  each  scarification  an  area  of  about  3-4  square 
centimeters.  Into  these  areas  the  virus  is  rubbed,  using  for  each 
small  area  a  quantity  about  sufficient  to  vaccinate  three  children. 
Two  to  three  centimeter  spaces  are  left  between  the  lesions.  The 
lesions  are  then  allowed  to  dry  and  may  be  covered  with  sterile 
gauze  or,  as  in  Vienna,  with  a  paste  made  up  of  beeswax,  gum  arabic, 
zinc  oxid,  water,  and  glycerin.  In  some  institutes  the  lesions  are 
left  entirely  uncovered. 

Ordinarily  within  about  twenty-four  hours  after  vaccination  a 
narrow  pink  areola  appears  about  the  scratches.  Within  forty-eight 

14  Park  and  Williams,  Path.  Microorg.,  N.  Y.,  1914,  p.  569. 


GENERAL   CONSIDERATION   OF   FILTRABLE   VIRUS  897 

hours  the  scratches  themselves  become  slightly  raised  and  papular, 
and  within  four  or  six  days  typical  vaccina  vesicles  have  usually 
developed. 

To  obtain  the  vaccine  from  such  lesions,  the  entire  operative 
field  is  carefully  washed  with  warm  water  and  soap,  followed  by 
sterile  water.  In  some  cases  two  per  cent  lysol  is  employed,  but 
must  again  be  thoroughly  removed  by  subsequent  washing  with 
sterile  water.  Crusts,  if  present,  are  then  carefully  picked  off  and 
the  entire  contents  of  the  vesicle,  sticky  serum,  and  pulpy  exudate 
removed  by  the  single  sweep  of  a  spoon-curette.  The  curetted 
masses  are  caught  in  sterile  beakers  or  tubes  and  to  them  is  added 
four  times  their  weight  of  a  mixture  of  glycerin  fifty  parts,  water 
forty-nine  parts,  and  carbolic  acid  one  part.15  German  workers 
prefer  a  mixture  of  glycerin  eighty  parts,  and  water  twenty  parts, 
omitting  the  use  of  carbolic  acid.  The  glycerinated  pulp  is  allowed 
to  stand  for  three  or  four  weeks  in  order  to  allow  bacteria,  which 
are  invariably  present,  to  die  out.  After  preservation  for  such  a 
length  of  time,  moreover,  thorough  emulsification  is  obtained  more 
easily  than  when  this  is  attempted  immediately  after  curettage.  At 
the  end  of  three  or  four  weeks,  the  glycerinated  pulp  is  thoroughly 
triturated,  either  with  mortar  and  pestle  or  by  means  of  specially 
constructed  triturating  devices.  Pulp  so  prepared  should  remain 
active  for  at  least  three  months  if  properly  preserved  in  sealed  tubes 
in  a  dark  and  cool  place. 

From  the  serum  oozing  from  the  bases  of  the  lesions,  after  curet- 
tage, bone  or  ivory  slips  may  be  charged  for  vaccination  with  dry 
virus.  The  glycerinated  pulp  is  put  up  in  small  capillary  tubes, 
sealed  at  both  ends,  and  distributed  in  this  form.  Park  states  that 
a  calf  should  yield  about  10  grams  of  pulp  (which  when  made  up 
should  suffice  to  vaccinate  1,500  persons),  and.  in  addition  about 
200  charged  bone  slips. 

The  virus  may  be  tested  for  its  efficiency  by  a  variety  of  methods. 
Calmette  and  Guerin  inoculate  rabbits  upon  the  inner  surfaces  of 
the  ears  and  estimate  the  potency  of  the  virus  from  the  speed  of 
development  and  extensiveness  of  the  resulting  lesions.  Guerin16 
has  estimated  the  potency  of  virus  quantitatively  by  a  method 
depending  upon  the  inoculation  of  rabbits  with  a  series  of  dilutions. 


™Huddleston,  quoted  in  Park,  "Pathogenic  Bacteria/'  N.  Y.    1908. 
18  Guerin,  Ann.   de  1'inst.  Pasteur,   1905. 


898  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

Beginning  with  a  mixture  containing  equal  weights  of  glycerin  and 
vaccine  pulp,  dilutions  are  made  with  sterile  water  ranging  from 
1  in  10  to  1  in  100.  Rabbits  are  shaved  over  the  skin  of  the  back 
and  1  c.c.  of  each  of  these  dilutions  is  rubbed  into  the  shaved  areas. 
Fully  potent  virus  should  cause  closely  approximated  vesicles  in  a 
dilution  of  1  in  500,  and  numerous  isolated  vesicles  in  a  dilution 
as  high  as  1  in  1,000. 

Quantitative  estimations  of  the  bacteria  in  the  glycerinated  virus 
should  be  made  by  the  plating  method  and  the  vaccine  used  only 
when  after  several  weeks  of  preservation  the  numbers  of  the  bacteria 
have  been  greatly  diminished.  In  glycerinated  pulp  the  bacteria 
will  often  disappear  entirely  in  the  course  of  a  month.  The  vaccine 
should  also  be  tested  for  the  possible  presence  of  tetanus  bacilli,  by 
the  inoculation  of  white  mice. 

Vaccination  of  human  beings  is  performed  by  slightly  scarifying 
the  skin  of  the  arm  or  leg  with  a  sharp  sterile  needle  or  lancet  and 
rubbing  into  the  lesion  potent  vaccine  virus.  The  virus  was  formerly 
dried  upon  wood,  bone,  or  ivory  slips  and  moistened  with  sterile 
water  before  the  operation.  At  the  present  day  the  glycerinated 
pulp  is  almost  universally  employed. 

Since  the  ordinary  scarification  method  has  not  been  universally 
satisfactory,  other  methods  of  vaccination  have  been  suggested. 
Occasional  failure  of  the  scarification  method  may  in  part  be  due 
to  the  fact  that  the  glycerinated,  ripened  virus  as  used  in  most 
countries,  has  lost  considerably  in  potency.  We  have  seen  men  in 
the  American  Army  successfully  vaccinated  with  French  virus  after 
two  vaccinations  with  American  virus  had  failed.  The  French  virus 
used  had  not  been  allowed  to  ripen  in  glycerine,  was  reasonably 
fresh  (so-called  green  virus)  and  was,  therefore,  probably  more 
potent.  The  fact  that  it  still  contained  staphylococci  and  other 
organisms  did  not  in  these  cases  lead  to  infections  of  any  importance. 
It  must,  however,  always  be  dangerous,  and  the  use  of  green  virus, 
while  perhaps  more  efficient  from  the  point  of  view  of  vaccination, 
does  not  seem  to  us  to  be  commendable.  It  is  probably  better  to 
attempt  modification  of  the  present  method  by  a  more  efficient  in- 
troduction of  the  virus.  The  alternative  methods  which  have  been 
suggested  are  puncture  in  which  drops  of  virus  are  placed  on  the 
skin  and  punctures  made  through  the  drops.  Scarification  or  small 
incisions  through  the  drops  have  also  been  recommended.  Recent 


GENERAL   CONSIDERATION   OF   FILTRABLE   VIRUS          899 

studies  by  Wright17  have  shown  that  the  intracutaneous  injection 
of  0.1  c.c.  of  the  ordinary  glycerinated  virus  diluted  with  one  part 
of  sterile  distilled  water,  resulted  in  " takes"  when  the  ordinary 
scarification  methods  were  unsuccessful.  He  vaccinated  227  negro 
soldiers  in  this  way,  and  obtained  over  70  per  cent  successful  vac- 
cinations when  only  slightly  over  8  per  cent  were  obtained  on  the 
same  series  of  cases  previously,  by  the  incision  method. 

Sternberg,  Kinyoun  and  others  have  demonstrated  that  within 
two  weeks  after  vaccination  the  blood  serum  of  the  vaccinated 
patients  will  neutralize  vaccine  virus  if  allowed  to  stand  with  it 
in  a  test  tube  overnight. 

That  vaccination  is  of  incalculable  benefit  to  the  human  race  is 
no  longer  a  question  of  opinion,  and  opposition  to  the  practice  is 
explicable  only  on  the  basis  of  ignorance.  Statistical  compilations 
upon  this  point  are  very  numerous.  It  may  suffice  to  select  from 
the  voluminous  literature  a  single  example,  taken  from  Jiirgensen, 
which  embodies  the  statistics  of  death  from  smallpox  in  Sweden, 
during  the  periods  immediately  preceding  and  following  the  intro- 
duction of  vaccination*  In  that  country  the  first  vaccination  was 
done  in  1801.  By  1810  the  practice  was  generally  in  use  but  not 
enforced.  In  1816  it  was  legally  enforced.  The  years  from  1774 
to  1855  can  thus  be  divided  into  three  periods. 


1.  Prevaccinal  period,  1774-1801  (25  years).     Deaths  smallpox  per 

million  inhabitants   2,050 

2.  Transitional  period,  1801-1810   (9  years) 680 

3.  Vaccination  enforced,  1810-1855  (35  years) 169 

Prevaccinal  period  death  rate  20.00  per  mille. 
Vaccinal  period  death  rate          0.17  per  mille. 

In  considering  the  benefit  of  vaccination  it  must  not  be  forgotten 
that  revaccination  is  quite  as  important  as  the  first  vaccination, 
which  confers  immunity  only  for  from  seven  to  ten  years.  A  child 
should  therefore  be  vaccinated  soon  after  birth  or  at  least  before 
the  eighth  month,  and  the  process  should  be  repeated  about  every 
seven  years  thereafter. 


17  Wright,  Jour.  A.  M.  A.  71,  1918,  654. 


900  DISEASES  CAUSED  BY   F1LTRABLE   VIRUS 

RABIES 

(Hydrophobia,  Rage,  Lyssa,  HundswutJi) 

Rabies  is  primarily  a  disease  of  animals,  infectious  for  practically 
all  the  mammalia,  but  most  prevalent  among  carnivora,  dogs,  cats, 
and  wolves.  It  is  said  also  to  occur  spontaneously  among  skunks 
of  the  Southwestern  United  States,  and  is  readily  inoculable  upon 
guinea-pigs,  rabbits,  mice,  rats,  and  certain  birds,  chicken  and  geese 
being  especially  susceptible.  Man  is  subject  to  the  disease.  Infec- 
tion usually  occurs  as  a  consequence  of  the  saliva  of  rabid  animals 
gaining  entrance  to  wounds  from  bites  or  scratches.  The  disease 
is  more  or  less  widely  prevalent  in  all  civilized  countries  except 
England,  where  the  careful  supervision  of  dogs,  enforcement  of 
muzzling  laws,  and  rigid  legislation  regarding  the  importation 
of  dogs,  have  caused  a  practical  eradication  of  the  disease. 
A  fair  estimate  of  the  prevalence  of  the  disease  may  be  ob- 
tained from  the  statistics  -  of  animals  dyin^  or  killed  because  of 
rabies  in  different  countries.  In  Germany,  according  to  Kolle  and 
Hetsch,  during  the  fifteen  years  ending  in  1901,  there  were  9,069 
dogs,  1,664  cattle,  191  sheep,  110  horses,  175  hogs,  79  cats,  16  goats, 
1  mule,  and  1  fox  affected  with  rabies.  In  eastern  United  States 
the  disease  is  not  uncommon.  The  statistics  of  the  New  York 
Department  of  Health,  for  a  period  of  six  months  ending  December 
31,  1907,  show  seventy-four  cases  of  rabies  among  dogs  in  New 
York  City  and  vicinity.  Among  human  beings  the  disease  is  no 
longer  common  in  civilized  countries,  since  early  preventive  treat- 
ment is  successfully  applied  in  almost  all  infected  subjects. 

Experimental  infection  in  susceptible  animals  is  best  carried  out 
by  injections  of  a  salt-solution  emulsion  of  the  brain  or  spinal  cord 
of  an  afflicted  animal,  subdurally,  through  a  trephined  opening  in 
the  skull,  but  may  also  be  accomplished  by  injection  into  the  per- 
ipheral nerves,  the  spinal  canal,  or  the  anterior  chamber  of  the  eye, 
Intravenous  and  intramuscular  injections  are  also  successful,  though 
less  regularly  so. 

The  time  of  incubation  after  inoculation  varies  with  the  nature 
of  the  virus  used,  the  location  of  the  injection,  and  the  quantity 
injected.  In  accidental  infections  of  man  and  animals  the  incuba- 
tion is  shortest  and  the  disease  most  severe  when  the  wounds  are 


GENERAL   CONSIDERATION   OF   FILTRABLE   VIRUS  901 

about  the  head,  neck,  and  upper  extremities  and  are  deeply  lacerated. 
This  is  explained  by  the  fact  that  the  poison  is  conveyed  to  the 
central  nervous  system  chiefly  by  the  path  of  the  nerve  trunks. 
This  has  been  experimentally  shown  by  di  Vcstca  and  Zagari18  who 
inoculated  animals  by  injection  into  peripheral  nerves,  and  showed 
that  the  nerve  tissue  near  the  point  of  inoculation  becomes  infectious 
more  quickly  than  the  parts  higher  up;  thus  the  lumbar  cord  of 
an  animal  inoculated  in  the  sciatic  nerve  is  infectious  several  days 
before  virus  can  be  demonstrated  in  the  medulla. 

In  man,  infected  with  "street  virus,"  that  is,  with  the  virus  of 
a  dog  or  other  animal  not  experimentally  inoculated,  the  incubation 
period  varies  from  about  forty  to  sixty  days.  Isolated  cases  have 
been  reported  in  which  this  period  was  prolonged  for  several  months 
beyond  this. 

The  virulence  of  rabic  virus  may  be  markedly  increased  or 
diminished  by  a  number  of  methods.  By  repeated  passage  of  the 
virus  through  rabbits,  Pasteur19  was  able  to  increase  its  virulence 
to  a  more  or  less  constant  maximum.  Such  virus  which  had  been 
brought  to  the  highest  obtainable  virulence,  he  designated  as  "virus 
fixe."  Inoculation  of  rabbits,  dogs,  guinea-pigs,  rats,  and  mice  with 
such  virus  usually  results  in  symptoms  within  six  to  eight  days. 
The  same  animals  inoculated  with  street  virus  may  remain  ap- 
parently healthy  for  two  to  three  weeks. 

In  dogs  and  guinea-pigs  inoculation  usually  results  first  in  a  stage 
of  increased  excitability,  restlessness,  and  sometimes  viciousness. 
This  is  followed  by  depression,  torpor,  loss  of  appetite,  inability 
to  swallow,  and  finally  paralysis.  In  rabbits  the  disease  usually 
takes  the  form  of  what  is  known  as  "dumb  rabies,"  the  animals 
gradually  growing  more  somnolent  and  weak,  with  tremors  and 
gradual  paralysis  beginning  in  the  hind  legs. 

In  experimentally  infected  birds  the  disease  is  slow  in  appearing 
and  may  show  a  course  of  gradually  increasing  weakness  and 
progressive  paralysis  extending  over  a  period  of  two  weeks  after 
the  appearance  of  the  first  symptoms. 

In  man,  the  disease  begins  usually  with  headaches  and  nervous 
depression.  This  is  followed  by  difficulty  in  swallowing  and  spasms 
of  the  respiratory  muscles.  These  symptoms  occur  intermittently, 

18  di  Vested  and  Zagari,  Ann.  de  1'inst.  Pasteur,  iii. 

19  Pasteur's  work  on  rabies.     Compt.  rend,  de  1'acad.  des  sciences,  1881,  1882, 
1884,  1885,  1886,  and  Ann.  de  1'inst.  Pasteur,  1887  and  1888. 


902  DISEASES  CAUSED  BY   FILTRABLE  VIRUS 

the  free  intervals  being  marked  by  attacks  of  terror  and  nervous 
depression.  Occasionally  there  are  maniacal  attacks  in  which  the 
patient  raves  and  completely  loses  self-control.  Finally,  paralysis 
sets  in,  ending  eventually  in  death. 

Pathological  examination  of  the  tissues  of  rabid  animals  and 
human  beings  reveals  macroscopically  nothing  but  ecchymoses  in 
some  of  the  mucous  and  serous  membranes.  Microscopically,  how- 
ever, many  abnormal  changes  have  been  observed  and  were  formerly 
utilized  in  histological  diagnosis  of  the  condition.  Babes20  has 
described  a  disappearance  of  the  chromatic  element  in  the  nerve 
cells  of  the  spinal  cord.  This  observation  has  been  confirmed  by 
others,21  but  is  no  longer  regarded  as  pathognomonic  of  rabies.  The 
same  observer  has  described  a  marked  leucocytic  infiltration  which 
occurs  about  the  blood-vessels  of  the  brain  and  about  the  ganglia 
of  the  sympathetic  system.  These  changes  are  not  found  in  animals 
infected  with  virus  fixe  and  are  present  only  in  animals  and  human 
beings  inoculated  with  street  virus. 

In  1903  Negri22  of  Pavia  described  peculiar  structures  which  he 
observed  in  the  cells  of  the  central  nervous  system  of  rabid  dogs. 
While  present  in  all  parts  of  the  brain,  these  " Negri  bodies"  are 
most  regularly  present  and  numerous  in  the  larger  cells  of  the 
hippocampus  major  and  in  the  Purkinje  cells  of  the  cerebellum- 
The  presence  of  these  structures  in  rabid  animals  and  man  has 
been  confirmed  by  a  large  number  of  workers  in  various  parts  of 
the  world,  and  the  specific  association  of  these  bodies  with  the 
disease  is  now  beyond  doubt.  In  consequence,  the  determination 
of  "Negri  bodies"  in  the  brains  of  suspected  animals  has  become 
an  extremely  important  method  of  diagnosis — more  rapid  and  ac- 
curate than  the  methods  previously  known. 

The  demonstration  of  Negri  bodies  in  tissues  is  carried  out  as 
follows:  A  small  piece  of  tissue  is  taken  from  the  cerebellum  or 
from  the  center  of  the  hippocampus  major  (cornu  ammonis),  and 
is  fixed  for  twelve  hours  in  Zenker's  fluid.  It  is  then  washed 
thoroughly  in  water  and  dehydrated  as  usual  in  graded  alcohols, 
embedded  in  paraffin,  and  sectioned.  The  sections  are  best  stained 
by  the  method  of  Mann,  as  follows: 


30  Babes,  Virch.  Arch.,  110,  and  Ann.  de  Pinst.  Pasteur,  6,  1892. 
nVan  Gehuchten,  Bull,  de  1'aead.  de  med.  et  biol.,  1900. 
"Negri,  Zeit.  f.  Hyg.,  xliii  and  xliv. 


GENERAL   CONSIDERATION   OF   FILTRABLE   VIRUS  903 

The  sections,  attached  to  slides  in  the  usual  way,  are  immersed  in  the 
following  solution  for  from  twelve  to  twenty-four  hours : 

Methylene-bluc  (Gruebler  OO),  1  per  cent 35  c.c. 

Eosin   (Gruebler  BA),  1  per  cent 35  c.c. 

Distilled  water 100  c.c. 

They  are  then  differentiated  in: 

Absolute  alcohol    30  c.c. 

Sodium  hydrate,  1  per  cent  in  absolute  alcohol 5  c.c. 

In  this  solution  blue  is  given  off  and  the  sections  become  red.  After  about 
five  minutes  the  sections  are  removed  from  this  solution,  are  washed  in 
absolute  alcohol,  and  are  placed  in  water  where  they  again  become  faintly 
bluish.  It  is  of  advantage  to  immerse  them,  now,  in  water  slightly  acidified 
with  acetic  acid.  Following  this  they  are  dehydrated  with  absolute  alcohol 
and  cleared  in  xylol,  as  usual. 

In  preparations  made  in  this  way,  the  nerve  cells  are  stained  a 
pale  blue,  and  in  their  -cytoplasm,  lying  either  close  to  the  nucleus 
or  near  the  root  of  the  axis-cylinder  process,  are  seen  small  oval 
bodies  stained  a  deep  pink.  The  bodies  are  variable  in  size,  measur- 
ing from  1  to  27  micra  in  diameter.  They  are  round  or  oval,  show 
a  more  deeply  stained  peripheral  zone  which  has  been  interpreted 
as  a  cell  membrane,  and,  in  their  interior,  often  show  small  vacuole- 
like  bodies.  There  may  be  more  than  one,  often  as  many  as  three 
or  four,  in  a  single  cell. 

The  rapid  demonstration  of  Negri  bodies  in  smears  of  brain 
tissue  has  recently  been  advocated  by  many  observers  and  has  been 
extensively  used  for  diagnosis.  It  is  carried  out,  according  to  Van 
Gieson,23  in  the  following  way :  A  small  pin-head-sized  piece  of  brain 
tissue  from  the  regions  indicated  above,  is  placed  on  one  end  of 
a  slide  under  a  cover-glass  and  the  cover  is  gently  squeezed  with 
the  finger  until  the  tissue  is  flattened  out  into  a  thin  layer.  The 
glass  cover  is  then  gently  shifted  across  the  slide  until  the  brain 
tissue  is  smeared  along  the  entire  surface.  These  smears  may  be 
fixed  in  methyl  alcohol  and  stained  by  the  Giemsa  method,  as 
described  in  the  chapter  on  Spirochaeta  pallida. 

Stained  in  this  way,  the  Negri  bodies  are  stained  light  blue,  in 
contrast  to  Hie  darker  and  more  violet  cell-bodies. 

28  Van  Gieson,  Proc.  of  N.  Y.  Pathol.  Soc.,  N.  S.,  iv,  1906, 


904  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

The  smears  may  also  be  stained  by  a  method  originated  by  Van 
Gieson,  which  gives  an  excellent  contrast  stain  and  reveals  more 
clearly  the  inner  structure  of  the  Negri  bodies.  Van  Gieson 's  stain 
is  prepared  as  follows: 

Distilled  water    10  c.c. 

Saturated  alcoholic  solution  of  rosanilin  violet 2  drops. 

Saturated  aqueous  solution  of  methylene-blue  diluted  one-half 

with  water 2  drops. 

This  method  has  been  modified  by  Williams  and  Lowden,24  who  add 
to  10  c.c.  of  distilled  water  three  drops  of  saturated  alcoholic  basic 
fuchsin  and  2  c.c.  of  Loeffler's  methylene-blue.  The  slides  are  fixed 
in  methyl  alcohol,  washed  in  water,  and  covered  with  the  freshly 
prepared  stain.  The  slide  is  held  over  the  flame  until  the  solution 
steams  and  is  then  rinsed  in  water  and  dried.  The  Negri  bodies 
assume  a  brilliant  red  and  contain  in  their  interior  darkly  stained, 
irregular  particles  which  have  been  interpreted  as  chromatin  bodies. 
As  to  the  nature  of  the  Negri  bodies  opinions  are  still  divided.  Their 
constant  presence  in  rabic  brain  tissue  is  unquestioned  and  their 
diagnostic  significance  well  established.  Cultivation  experiments, 
however,  have  been  uniformly  unsuccessful.  A  number  of  observers, 
Negri  himself,  Calkins,25  Williams  and  Lowden,26  and  others,  believe 
these  bodies  to  be  protozoa.  The  last-named  authors  base  this  opinion 
upon  the  definite  morphology  of  the  bodies,  and  their  staining 
properties,  which  in  many  respects  are  similar  to  those  of  protozoa. 
These  observers  also  claim  that  the  morphology  of  the  bodies  shows 
a  number  of  regular  cyclic  changes  which  are  found  accompanying 
different  stages  of  the  disease ;  these  changes  correspond,  according 
to  these  workers,  to  similar  cycles  occurring  among  known  protozoa 
of  the  suborders  of  the  class  Sporozoa.  Many  pathologists  still  look 
upon  them  as  specific  degenerations  of  the  nerve  cells  similar  to 
the  changes  observed  by  Babes. 

It  is  not  possible  to  decide  absolutely  from  the  facts  at  present 
at  our  disposal  whether  or  not  the  Negri  bodies  should  be  regarded 
as  parasites  or  as  specific  degeneration  products.  Their  constant 
presence  in  rabic  animals,  and  their  apparent  absence  from  normal 


24  Williams  and  Lowden,  Jour.  Inf.  Dis.,  3,  1906. 

25  Calkins,  Discussion,  Proc.  N.  Y.  Pathol.  Soc.,  N.  S.,  vol.  vi,  1906. 
56  Williams  and  Lowden,  loc.  cit. 


GENERAL   CONSIDERATION   OF   FILTRABLE    VIRUS  905 

brains  and  the  brains  of  animals  dead  of  other  diseases,  would  tend 
to  favor  the  parasitic  view.  To  us  it. would  seem  that  added  to 
this  the  clear  outlines,  apparent  regularity  of  structure,  and  curiously 
consistent  grouping  of  the  darkly  staining  inclusions  would  add 
weight  to  such  an  assumption.  We  have  triturated  rabic  tissue 
and  shaken  it  up  in  anti-formin  and  seen  many  free  Negri  bodies 
apparently  enucleated  from  the  cells  in  consequence.  Such  com- 
plete extrusion  from  the  cell  also  is  seen  in  the  ordinary  smear 
preparations.  It  is  at  least  unlikely  that  a  cell-degeneration  area 
would  be  expelled  from  the  cytoplasm  in  so  clearly  outlined  and 
morphologically  unaltered  a  form.  The  fact  that  the  virus  is  filtrable, 
as  shown  by  Remlinger,27  Poor  and  Steinhardt,28  and  others,  would 
on  the  other  hand  seem  to  contradict  the  etiological  importance  of 
the  Negri  bodies  unless,  with  some  of  the  observers  named,  we 
assumed  them  to  represent  a  large  stage  in  the  life-cycle  of  a 
protozoan  parasite,  which  also  occurred  in  smaller  forms.  It  is  a 
curious  fact,  also,  that  Negri  bodies  are  scarce  or  absent  in  the 
spinal  cord  and  cerebrum,  though  these  areas  are  as  virulent  or 
more  so  than  the  hippocampus  and  cerebellum.  They  are  small  and 
hard  to  find  in  virus  fixe,  largest  and  most  plentiful  in  cases  in  which 
the  incubation  period  has  been  prolonged — as  with  street-virus  in- 
fection. Much  can  be  said  on  both  sides,  but  in  analyzing  the  present 
experimental  facts,  it  seems  fair  to  say  that  neither  point  of  view 
is  certain,  though  the  parasitic  nature  of  the  Negri  bodies  seems 
very  likely. 

The  cultivation  of  parasites  from  rabic  tissues  has  of  course  been 
attempted  by  most  bacteriologists  who  have  studied  the  disease  since 
Pasteur.  In  all  attempts,  until  very  recently,  either  no  results  were 
obtained  or  else  the  parasites  described  could  be  shown  to  be  present 
because  of  extraneous  contamination.  Recently  Noguchi  announced 
that  he  has  been  able  to  cultivate  the  virus  by  employing  a  technique 
similar  to  his  methods  of  cultivating  Treponema  pallidum  and 
poliomyelitis  virus.  Into  high  tubes  filled  with  ascitic  fluid  a  bit  of 
fresh  sterile  rabbit  kidney  and  a  small  piece  of  rabic  virus  were 
placed.  The  ascitic  fluid  was  covered  with  sterile  oil  and  the  tubes 
incubated  at  37.5°  C.  After  five  days'  incubation  cloudiness  ap- 
peared and  with  it,  minute  globoid  bodies  not  unlike  those  seen  in 


^Eemlinger,  Ann.  de  Pinst.  Past.,  xvii,   1903. 

28  Poor  and  tfteinltardt,  Jour,  of  Inf.  Dis.,  xii,  1913. 


906  DISEASES  CAUSED  BY  FILTRABLE   VIRUS 

poliomyelitis.  After  several  generations  large  highly  refractile 
bodies  with  dark  central  spots  appeared  in  the  cultures,  and  these 
Noguchi29  regards  as  possibly  the  parasites  and  similar  to  Negri 
bodies.  Opinions  are  still  divided  as  to  the  significance  of  Noguchi's 
results.  However,  whatever  may  be  one's  opinion  regarding  the 
nature  of  the  peculiar  bodies  visible  in  his  cultures,  he  has  accom- 
plished the  feat  of  preserving  the  virulence  of  the  virus  through 
twenty-one  generations  on  artificial  media,  a  fact  which  alone  would 
seem  to  prove  that  he  had  successfully  cultivated  it,  even  though  his 
" nucleated  bodies"  do  not  eventually  turn  out  to  be  anything  more 
than  cell  degenerations.  The  possibility  that  he  may  have  carried 
original  virus  through  twenty-one  generations  and  that  it  has 
remained  virulent  for  about  100  days  at  37.5°  C.  can  not  be  excluded 
as  yet,  but  seems  very  remote. 

The  Specific  Therapy  of  Rabies.— The  treatment  which  is  now 
prophylactically  applied  to  patients  infected  with  or  suspected  of 
infection  with  rabies  has  been  but  little  altered  either  in  principle 
or  in  technical  detail  since  it  was  first  worked  out  by  Pasteur.  In 
principle  it  consists  of  an  active  immunization  with  virus,  attenuated 
by  drying,  administered  during  the  long  incubation  period  in  doses 
of  progressively  increasing  virulence. 

By  the  repeated  passage  of  street  virus  through  rabbits,  Pasteur 
obtained  a  virus  of  maximum  and  approximately  constant  virulence 
which  he  designated  as  virus  fixe.  By  a  series  of  painstaking  experi- 
ments he  then  ascertained  that  such  virus  fixe  could  be  gradually 
attenuated  by  drying  over  caustic  potash  at  a  temperature  of  about 
25°  C.,  the  degree  of  attenuation  varying  directly  with  the  time  of 
drying.  Thus,  while  fresh  virus  fixe  regularly  caused  death  in  rabbits 
after  six  to  seven  days,  the  incubation  time  following  the  inoculation 
of  dried  virus  grew  longer  and  longer  as  the  time  of  drying  was 
increased,  until  finally  virus  dried  for  eight  days  was  no  longer 
regularly  infectious  and  that  dried  for  twelve  to  fourteen  days  had 
completely  lost  its  virulence. 

The  method  of  active  immunization  which  Pasteur  used  consisted 
in  injecting,  subcutaneously,  virus  of  progressively  increasing  viru- 
lence, beginning  with  that  derived  from  cords  dried  for  thirteen  days 
and  gradually  advancing  to  a  strong  virus.  Thus  the  patient  was 
immunized  to  a  potent  virus  several  weeks  before  the  incubation  time 

28  Noguchi,  Jour.  Exp.  Med.,  xviii,  1913. 


GENERAL   CONSIDERATION    OF    FILTRABLE    VIRUS 


907 


of  his  own  infection  had  elapsed.  Pasteur  successfully  proved  the 
efficacy  of  his  method  upon  dogs  and  finally  upon  human  beings,  the 
first  human  case  being  that  of  a  nine-year-old  child — Joseph  Meister. 
TECHNIQUE  OF  RABIES  THERAPY. — The  technique  developed  by  Pas- 
teur is  still,  in  the  main,  followed  by  those  who  treat  rabies  to-day. 

I.  As  a  preliminary,  it  is  necessary  to  prepare  or  obtain  virus  fixe. 
This  may  generally  be  procured  from  an  established  laboratory  or 
may  be  prepared  independently  by  passing  street  virus  through  a 
series  of  young  rabbits  (weighing  from  700  to  1,000  gms.).    According 
to  Hogyes,30  the  passage  of  the  virus  through  twenty-one  to  thirty 
rabbits,  in  this  way,  will  reduce  its  incubation  time 

to  seven  or  eight  days.  Babes  claims  to  obtain  a  virus 
fixe  more  rapidly  by  passing  the  virus  alternately 
through  rabbits  and  guinea-pigs. 

For  purposes  of  inoculation,  virus  is  prepared  by 
emulsifying  in  sterile  salt  solution  pieces  of  the 
medulla  or  cerebellum  of  animals  dead  of  a  previous 
inoculation.  The  brain  tissue  which  is  not  emulsified 
may  be  preserved  under  sterile  glycerin  in  a  dark  and 
cool  place  for  further  use. 

II.  Rabbits  are  inoculated  with  virus  fixe  by  in- 
tracranial  injection.    A  small  incision  is  made  in  the 
shaved   scalp    in    the   median   line,    and   the   skin   is 


SPINAL  CORD  OF 
RABBIT  FOR 
PURPOSES  OF 
ATTENUATION. 


retracted.     With  a  small  trephine  or  a  round  chisel,  FIG.  100. — 

an  opening  is  made  in  the  skull   and  in  the   angle      OD  OF  DRYING 

between  the  coronary  and  sagittal  sutures.     Through 

this  opening  about  0.2  to  0.3  c.c.  of  the  virus  fixe  is 

injected,  either  directly  into  the  brain  substance  or 

simply  under  the  dura. 

As  soon  as  a  rabbit  so  inoculated  has  died  it  is  autopsied.  The 
animal  before  dissection  should  be  washed  in  a  disinfectant  solution 
— lysol  or  carbolic  acid.  The  skin  is  then  removed  and  the  animal, 
lying  on  its  ventral  surface,  is  fastened  to  a  dissecting  board.  The 
spinal  canal  is  then  laid  open  with  a  pair  of  curved  scissors  and 
the  spinal  cord  carefully  removed.  This  is  accomplished  by  cutting 
across  the  cord  in  the  lumbar  region,  and  lifting  this  with  a  forceps 
while  the  nerve  roots  are  divided  from  below  upward. 

Tlic  cord  is  suspended  by  a  sterile  thread  within  a  large  bottle 


*°  Hogyes,  quoted  from  Kraus  and  Levaditi,  "Handb., "  etc.,  I. 


908 


DISEASES   CAUSED   BY   F1LTHABLE   VIRUS 


into  the  bottom  of  which  pieces  of  potassium  hydrate  have  been 
placed.  The  bottle  is  then  set  away  in  a  dark  room  or  closet,  the 
temperature  of  which  is  regulated  so  as  to  vary  little  above  25°  C. 
Bacteriological  controls  as  to  the  sterility  of  the  cord  should  also 
be  made. 

After  drying,  pieces  of  the  cord  are  prepared  for  injection.  This 
is  done  in  various  ways.  At  the  New  York  Department  of  Health  1  cm. 
of  the  cord  is  emulsified  in  3  c.c.  of  sterile  salt  solution,  the  dose  for 
injection  being  usually  2.5  c.c.  Marx31  emulsifies  1  cm.  of  the  cord  in 
5  c.c.  of  sterile  bouillon  or  salt  solution,  using  1  to  3  c.c.  of  this  for 
injection  according  to  the  age  of  the  cord.  For  shipment  20  per  cent 
of  glycerin  and  0.5  per  cent  of  carbolic  acid  arc  added. 

The  scheme  of  treatment  is  also  subject  to  variations  according 
to  the  individual  customs  of  various  laboratories.  The  following 
scheme  is  the  routine  of  the  Pasteur  Institute  in  Paris,  as  quoted 
in  Kraus  and  Levaditi,  ' '  Handbuch  f iir  Immunitatsf orschung, ' '  Vol. 
I,  p.  713. 


Day  of 
Treatment 

MILD  CASES 

Dose 

MEDIUM  CASES 

Dose 

SEVERE  CASES 

Dose 

Days  of 
Drying 

Days  of 
Drying 

Days  of  Drying 

1 

14  +  13 

3  C.C. 

14  +  13 

3  c.c. 

A.M.  14  +  13  P.M.  12  +  11 

3  c.c. 

2 

12  +  11 

3  C.C. 

12  +  11 

3  c.c. 

A.M.  10+    9P.M.     8+    7 

3  c.c. 

3 

10+  9 

3  c.c. 

10+  9 

3  c.c. 

A.M.           7        P.M.     6 

2  c.c. 

4 

8+  7 

3  c.c. 

8+  7    . 

3  c.c. 

5 

2  c.c. 

5 

6+  6 

3  c.c. 

6+  6 

3  c.c.  • 

5 

2  c.c. 

6 

5 

Ic.c. 

5 

2  c.c. 

4 

2  c.c. 

7 

5 

1  c.c. 

5 

2  c.c. 

3 

1  c.c. 

8 

4 

1  c.c. 

4 

2  c.c. 

4 

2  c.c. 

9 

3 

Ic.c. 

3 

1  c.c. 

3 

1  c.c. 

10 

5 

2  c.c. 

5      , 

2  c.c. 

5 

2  c.c. 

11 

5 

2  c.c. 

5 

2  c.c. 

5 

2  c.c. 

12 

4 

2  c.c. 

4 

2  c.c. 

4 

2  c.c. 

13 

4 

2  c.c. 

4 

2  c.c. 

4 

2  c.c. 

14 

3 

2  c.c. 

3 

2  c.c. 

3 

2  c.c. 

15 

3 

2  c.c. 

3 

2  c.c. 

3 

2  c.c. 

16 

i      5 

2  c.c. 

5 

2  c.c. 

17 

4 

2  c.c. 

4 

2  c.c. 

18 

3 

2  c.c. 

3 

2  c.c. 

19 

5 

2  c.c. 

20 

4 

2  c.c. 

21 

3 

2  c.c. 

31  Marx,  Deut.  med.  Woch.,  1899,  1900. 


GENERAL   CONSIDERATION   OF   FILTRABLE   VIRUS 


909 


The  treatment  at  the  New  York  Department   of  Health  is  as 
follows: 


Day  of 
Treatment 

MILD  CASES 

Dose 

MEDIUM  CASES 

Dose 

SEVERE  CASES 

Dose 

Days  of 
Drying 

Days  of 
Drying 

Days  of  Drying 

1 

14  +  13 

4  C.C. 

10 

4  C.C. 

A.M.  10+9  P.M.        10+9 

4  c.c. 

2 

12  +  11 

4  c.c. 

9 

4  c.c. 

A.M.     8+7  P.M.     8+7 

4  c.c. 

3 

10+9 

4  c.c. 

9 

4  c.c. 

6 

4  c.c. 

4 

8+7 

4  c.c. 

8+7 

4  c.c. 

4 

4  c.c. 

5 

6 

2  c.c. 

6 

2  c.c. 

3 

2  c.c. 

6 

5 

2  c.c. 

5 

2  c.c. 

4 

2  c.c. 

7 

4 

2  c.c. 

4 

2  c.c. 

3 

2  c.c. 

8 

3 

2  c.c. 

3 

2  c.c. 

2 

2  c.c. 

9 

5 

2  c.c. 

2 

2  c.c. 

4 

2  c.c. 

10 

4 

2  c.c. 

5 

2  c.c. 

1 

2  c.c. 

11 

3 

2  c.c. 

4 

2  c.c. 

4 

2  c.c. 

12 

5 

2  c.c. 

3 

2  c.c. 

3 

2  c.c. 

13 

4 

2  c.c. 

2 

2  c.c. 

2 

2  c.c. 

14 

3 

2  c.c. 

4 

2  c.c. 

4 

2  c.c. 

15 

5 

2  c.c. 

3 

2  c.c. 

1 

2  c.c. 

16 

4 

2  c.c. 

2 

2  c.c. 

4 

2  c.c. 

17 

4 

2  c.c. 

3 

2  c.c. 

18 

3 

2  c.c. 

2 

2  c.c. 

19 

2 

2  c.c. 

4 

2  c.c. 

20 

3 

2  c.c. 

21 

2 

2  c.c. 

22 

4 

2  c.c. 

23 

3 

2  c.c. 

24 

2 

2  c.c. 

25 

4 

2  c.c. 

26 

3 

2  c.c. 

The  severity  or  mildness  of  cases  is  estimated  from  the  depth 
and  degree  of  laceration  of  the  wounds,  also  from  their  location — 
bites  about  the  face  and  upper  extremities  b«ing  the  most  dangerous. 

During  the  course  of  such  treatment  patients  may  show  trouble- 
some erythema  about  the  point  of  injection  and  occasionally  back- 
ache and  muscular  pains.  Treatment  need  not  be  omitted  unless  these 
symptoms  become  excessive. 

The  efficiency  of  the  Pasteur  treatment  in  rabies  is  no  longer 
problematical.  According  to  Hogyes,  50,000  people  have  been 
treated  within  ten  years,  with  an  average  mortality  of  1  per  cent. 

Although  the  method  described  above  is  the  one  which  is  exten- 
sively used  in  all  established  institutes  for  the  treatment  of  rabies, 


910  DISEASES  CAUSED   BY   FILTRABLE  VIRUS 

other  methods  have  been  elaborated  and  used  to  a  slight  extent. 
One  of  the  most  important  of  these  is  the  "dilution  method"  of 
Hogyes.  This  method  is  carried  out  as  follows :  A  definite  quantity 
of  the  spinal  cord  of  a  rabbit  dead  of  virus  fixe  is  emulsified  in  100 
c.c.  of  normal  salt  solution.  Dilutions  of  this  emulsion  are  made 
and  the  patient  is  injected  at  first  with  a  dilution  of  1  -.1,000,  subse- 
quent injections  being  made  of  gradually  increasing  concentration 
until  a  concentration  of  1 :100  is  reached.  This  method,  so  far  as  it 
has  been  used,  has  been  satisfactory,  but  it  has  not  yet  found  exten- 
sive application. 

Harris  and  ShackelP2  describe  an  improved  method  of  desic- 
cating rabic  virus  which  consists  in  placing  the  material  to  be  dried 
in  the  bottom  of  a  vacuum  desiccating  jar  in  the  upper  part  of 
which  is  a  separate  dish  containing  sulphuric  acid.  The  temperature 
is  reduced  by  placing  the  jar  in  a  salt  and  ice  mixture,  and  after 
thorough  solidification  of  the  material  has  resulted,  a  rapid  vacuum 
is  produced  by  a  Geryk  pump  to  less  than  2  mm.  of  mercury.  The 
virus  so  dried  will  retain  its  virulence  for  as  long  as  four  months, 
if  guarded  against  moisture.  It  will  be  noted  that  this  method 
cannot  be  taken  as  justifying  any  particular  conclusions  as  to  the 
nature  of  the  rabic  virus  since  the  same  method  has  been  applied 
by  Swift  and  others  to  the  maintenance  of  the  virulence  of  bacteria. 

Harris33  believes  that  the  attenuation  of  a  rabic  cord  in  the 
Pasteur  method  does  not  depend  primarily  upon  the  loss  of  water, 
but  rather  upon  the  method  of  extracting  the  water.  Slow  desic- 
cation attenuates  the  virus,  Harris  concludes,  by  reason  of  the  con- 
centration of  salt  and  other  substances  in  solution  of  the  brain  and 
cord,  the  action  being  thus  essentially  a  chemical  one.  Harris  has 
studied  the  minimal  lethal  dose  of  his  rapidly  dried  material  on 
animals,  and  has  developed  with  this  material  a  modified  method 
of  immunizing  animals  and  human  beings  against  rabies. 

He  prepares  suspensions  (from  the  material  rapidly  dried  at 
zero  degrees  as  above)  by  emulsifying  10  milligrams  in  10  c.c.  salt 
solution.  This  gives  a  dilution  of  1  milligram  to  each  cubic  centi- 
meter of  emulsion,  and  from  this  basic  suspension  dilutions  are 
made. 

32  Harris  and  Shackell,  Jour,  of  Infec.  Dis.,  8,  1911,  47. 
83  Harris,  Jour,  of  Infec.  Dis.,  13,  1913,  155. 


GENERAL   CONSIDERATION   OF   F1LTRABLE    VIRUS  911 

He  tests  virulence  by  injecting  0.1  c.c.  of  the  dilution  into 
the  brain  of  a  rabbit,  by  trephining  and  passing  a  very  fine  hypo- 
dermic needle  through  the  brain  to  the  base  or  into  the  lateral 
ventricle,  so  that  none  of  the  material  may  escape  when  the  needle 
is  withdrawn.  He  finds  that,  by  this  method,  his  material  after 
preservation  of  three  weeks,  is  equivalent  to  that  of  fresh  cord. 
After  fifty  days  it  is  25  per  cent  more  infective  than  the  same  quan- 
tity of  the  "one  day"  cord  of  the  old  method.  After  200  days  its 
infectivity  is  exactly  equal  to  that  of  the  "two  day"  cord  of  the 
old  method.  After  500  days  it  is  two  and  one-half  times  as  infective 
as  the  "three  day"  cord.  The  only  precaution  that  is  absolutely 
essential  is  that  in  preparing  and  preserving  the  diluted  material, 
the  presence  of  moisture  must  be  absolutely  prevented. 

Having  prepared  this  material,  Harris  thought  it  worth  while  to 
follow  the  suggestion  of  Hogyes  who  has  developed  a  method  of 
immunization  dependent  upon  the  dilutions  of  virulent  virus,  in- 
stead of  using  the  Pasteur  method  of  quantitative  destruction  by 
drying.  Hogyes  had  treated  10,000  patients  by  his  method  without 
accident,  giving  from  70  to  220  M.  I.  D.,  or  minimal  infective  doses 
for  rabbits,  the  first  day  and  from  200  to  400  M.  I.  D.  on  the  second, 
the  M.  I.  D.  being,  as  in  Harris's  experiments,  determined  by  injec- 
tions into  the  brains  of  rabbits.  Harris  has  constructed  charts  from 
careful  experiments  in  which  he  tabulates  the  proportion  of  infec- 
tious to  non-infectious  material  in  a  milligram  of  desiccated  brain 
after  preservation  for  various  periods.  Unlike  Harvey  and  Mc- 
Kenderick  (quoted  from  Harris)  who  believe  that  when  rabic  virus 
has  lost  its  infectiousness,  it  no  longer  has  the  power  of  conferring 
immunity,  Harris  believes  that  when  all  the  infectivity  of  rabic 
material  has  been  destroyed  rapidly  by  light  and  various  tempera- 
tures, it  still  has  considerable  powers  of  conferring  immunity.  It  is, 
of  coarse,  impossible  to  determine  the  degree  of  immunity  conferred 
by  the  non-infective  portions  of  desiccated  material. 

Harris  begins  his  treatment  with  material  in  which  the  proportion 
of  living  to  non-infective  material  is  estimated  as  being  about  1  to 
25,  that  is,  material  at  least  six  months  old.  As  the  treatment 
continues,  he  gradually  increases  until  he  uses  material  which  con- 
tains 100  M.  I.  D.  per  milligram.  In  two  years  of  such  treatment, 
he  had  no  accidents  either  in  patients,  dogs  or  rabbits. 

Attempts  to  treat  active  rabies  with  the  sera  of  immunized 
animals  have  so  far  been  unsuccessful. 


CHAPTER  XL VI 

ACUTE  ANTEEIOE  POLIOMYELITIS 
LETHARGIC  ENCEPHALITIS 

THE  disease  known  as  acute  anterior  poliomyelitis  has  long  been 
recognized  as  an  acute  infectious  condition,  both  because  of  the  char- 
acteristics of  its  clinical  manifestations  and  of  its  epidemic  occur- 
rence. For  these  reasons  it  was  classified  with  acute  infectious  dis- 
eases by  Marie  and  by  Striimpell  long  before  any  experimental 
evidence  of  infection  was  obtained. 

Its  contagiousness,  while  not  a  proven  fact,  seemed  very  likely 
from  the  evidence  of  its  mode  of  spreading  and  has  been  removed 
from  the  sphere  of  mere  conjecture  by  the  careful  study  of  a  Swedish 
epidemic,  comprising  one  thousand  cases,  made  by  Wickman.1 

While  acute  anterior  poliomyelitis  is  almost  exclusively  a  disease 
of  childhood,  it  is  assumed  by  clinicians  that  it  is  etiologically 
closely  related  to,  possibly  identical  with,  certain  diseases  of  the 
adult,  characterized  by  bulbar  paralysis  and  acute  encephalitis.  Into 
this  category,  also,  some  observers  place  the  condition  known  as 
' '  Landry  's  paralysis. ' '  The  basis  for  the  identification  of  these  con- 
ditions with  poliomyelitis  lies  chiefly  in  the  similarity  of  the  pathol- 
ogical lesions  and  upon  the  fact  that  the  last-named  diseases  occur 
most  often  during  the  course  of  poliomyelitis  epidemics.  The  writer 
some  years  ago  obtained  a  typical  poliomyelitis  infection  in  a  monkey 
with  material  from  a  definite  case  of  Landry 's  paralysis  in  a  young 
woman. 

The  most  thorough  recent  clinical  study  of  acute  poliomyelitis  of 
which  we  know  is  the  one  by  Peabody,  Draper  and  Dochez.2  The 
incubation  period  of  the  disease  varies,  but  appears  to  be  about  10 
days.  Prodromal  symptoms,  if  they  appear  at  all,  come  on  just 
before  the  onset  of  the  disease.  They  may  be  very  mild,  consisting 

1  Wiclcman,  quoted  from  Landsteiner  and  Popper,  Zeit.  f.   Immunitatsforch., 
ii,  1909. 

2  Peabody,  Draper  and  Docliez,  Monog.  Eock  Inst.,  4,  Jan.,  1912. 

912 


ACUTE  ANTERIOR  POLIOMYELITIS  913 

of  slight  fever,  sweating,  drowsiness,  pain  in  the  neck  and  head,  and 
weakness.  Intestinal  symptoms  are  not  uncommon.  Again  cases 
may  begin  without  prodromal  symptoms  with  sudden  illness,  chill 
and  fever.  This  may  be  all  that  occurs  in  the  so-called  abortive  cases 
which  in  Wickman's  studies  represent  from  25  to  56  per  cent  of  all 
cases. 

During  the  early  periods  of  the  disease  the  cases  may  show  vari- 
ous types  of  development.  They  may  simply  show  signs  of  general 
infection,  they  may  resemble  influenza,  the  gastro-intestinal  symp- 
toms may  be  predominant,  and  others  may  show  signs  of  meningeai 
irritation.  The  neck  may  be  stiff,  but  not  with  the  involuntary  stiff- 
ness of  meningitis,  showing  rather  a  reflex  resistance  when  the  at- 
tempt is  made  to  move  the  head  forward.  Peabody,  Draper  and 
Dochez  state  that  the  best  appreciation  of  the  clinical  condition  in 
acute  poliomyelitis  may  be  had  by  considering  the  cases  as  (1)  the 
abortive  cases  which  never  become  paralysed,3  the  cerebral  group 
which  is  rare  and  in  which  involvement  of  the  upper  motor  neurons 
with  spastic  paralysis  is  the  chief  characteristic,  and  (2)  the  bulbo- 
spinal  group,  which  is  the  largest  group,  and  in  which  motor  neuron 
involvement  and  flaccid  paralysis  occur. 

In  the  pre-paralytic  stages  the  leucocytes  are  apt  to  be  slightly 
increased  in  number  and  there  is  a  definite  increase  of  the  poly- 
nuclears  by  10  or  15  per  cent.  A  leucocytosis  of  15,000  to  30,000 
is  stated  by  the  writers  mentioned  above  as  distinctly  suggestive  of 
the  disease,  especially  if  the  polynuclears  are  increased  at  the  ex- 
pense of  the  lymphocytes.  The  study  of  the  spinal  fluid  is  of  great 
value.  During  the  first  days  of  the  disease,  before  the  paralysis 
appears,  there  is  an  increase  of  the  cellular  contents  with  a  total 
which  may  run  as  high  as  500  cells  per  cubic  millimeter,  but  usually 
they  run  about  50  per  cubic  millimeter.  The  writers  above  quoted 
saw  two  cases  in  which  there  were  999  and  650  cells,  respectively. 
During  the  second  week,  of  45  cases  seen  by  them,  8  had  over  50,  and 
23  were  above  normal.  In  the  later  stages  of  the  disease  the  cell 
counts  come  back  to  normal.  During  this  early  stage,  most  of  the 
cells  consist  of  lymphocytes,  rarely  showing  predominant  poly- 
nuclears. The  globulin  contents  during  the  early  stages  are  prac- 
tically normal,  or  slightly  increased.  This,  however,  increases  later, 
as  the  cell  counts  drop.  The  fluid  reduces  Fehling's  solution. 

8  For  literature,  see  Landsteiner  and  Popper,  loc.  cit.  and  Wickman,  Die  Heine- 
Medinsche  Krankheit,  Berlin,  1911. 


914  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

ETIOLOGY 

An  important  advance  in  the  study  of  this  disease  was  made  in 
1908  when  Landsteiner  and  Popper  succeeded  in  transmitting  it  to 
two  monkeys  ( Cynocephalus  hamadryas  and  Macacus  rhesus).  The 
transmission  was  accomplished  by  intraperitoneal  injections  of  a  saline 
emulsion  of  the  spinal  cord  of  a  child  that  had  died  during  the  fourth 
day  of  an  attack  of  infantile  paralysis — during  the  stage  of  acute 
fever.  The  first  monkey  injected  became  severely  ill  six  days  after  the 
injection  and  died  on  the  eighth  day.  The  second  animal  became 
paralyzed  seventeen  days  after  the  injection  and  was  killed  two  days 
later.  Cultural  experiments  with  the  substance  injected  were  nega- 
tive, as  were  also  inoculation  experiments  carried  out  upon  guinea- 
pigs,  rabbits,  and  mice.  The  histological  lesions  produced  in  the  in- 
oculated monkeys  were  similar  to  those  occurring  in  children  afflicted 
with  the  disease. 

An  attempt  to  transmit  the  disease  to  another  monkey  with  spinal- 
cord  substance  of  the  animal  that  was  killed  resulted  negatively. 

Soon  after  the  successful  experiments  of  Landsteiner  and  Popper,  a 
similar  result  was  recorded  by  Knoepfelmacher.4  An  attempt  to  trans- 
mit the  disease  from  monkey  to  monkey  was  again  negative. 

Similar  positive  inoculation  results  were  published,  a  little  later 
than  this,  by  Flexner  and  Lewis5  in  November,  1909,  and  by  Strauss 
and  Huntoon  6  in  January,  1910. 

Flexner  and  Lewis,  in  their  work,  moreover,  succeeded  in  trans- 
mitting the  disease  through  several  inoculation- generations  of 
monkeys.  The  same  workers7  have  ascertained  that  inoculation  may 
be  successfully  applied  not  only  by  the  intraperitoneal  route  but  intra- 
cerebrally,  subcutaneously,  intravenously,  and  by  the  path  of  the  larger 
nerves.  They  also  proved  that  not  only  the  brain  and  cord  of  afflicted 
animals  contains  the  virus,  but  that  this  may  be  found,  during  the 
early  days  of  the  disease  at  least,  in  the  spinal  fluid,  the  blood,  the 
nasopharyngeal  mucosa,  and  lymph  nodes  near  the  site  of  inoculation. 

Landsteiner  and  Levaditi,8  meanwhile,   experimenting  with  the 


4  Knoepfelmacher,  Mediz.  Klinik,  v,  1909. 

8  Flexner  and  Lewis,  Jour.  Am.  Med.  Assn.,  53,  1909. 

•  Strauss  and  Huntoon,  N.  Y.  Med.  Jour.,  Jan.,  1910. 

1  Flexner  and  Lewis,  Jour.  Exp.  Med.,  12,  1909. 

•Landsteiner  and  Levaditi,  Comptes  rend,  de  la  soc.  de  biol.,  Nov.,  1909,  and 


ACUTE  ANTERIOR   POLIOMYELITIS  915 

virus  independently,  succeeded  in  transferring  the  disease  from  one 
animal  to  others,  demonstrated  that  the  virus  could  pass  through  the 
pores  of  a  Berkefeld  filter,  and  showed  that  the  virus  was  present  in 
the  salivary  glands — a  fact  which  may  prove  of  great  importance  in 
possibly  establishing  a  clew  to  the  mode  of  contagion  among  human 
beings.  The  same  authors,  as  well  as  Flexncr  and  Lewis,  were  able  to 
show  that  the  virus  was  preservable  under  glycerin  for  as  long  as  ten 
days  and  retained  its  virulence  for  from  seven  to  eleven  days  when 
dried.  What  has  been  said  concerning  infantile  paralysis  may  also  be 
taken  to  apply  to  Landry's  paralysis.  From  a  typical  case  of  Landry's 
paralysis  in  an  adult  the  writer  succeeded  in  obtaining  typical  poli- 
omyelitis in  monkeys  at  Stanford  University  in  1912.  The  clinical 
diagnosis  in  this  case  was  made  by  Wilbur.  The  disease  in  the  mon- 
keys was  typical  of  poliomyelitis  and  the  histological  sections  showed 
the  typical  lesions. 

According  to  Flexner  and  Lewis  the  virus  remains  active,  when 
frozen,  for  as  long  as  forty  days,  but  is  extremely  sensitive  to  heat, 
being  destroyed  by  a  temperature  of  from  45°  to  50°  C.  maintained  for 
thirty  minutes. 

Experiments  aimed  at  the  isolation  or  even  morphological  detection 
of  a  parasite  in  the  virulent  material  have  been  entirely  without  suc- 
cess until  recently.  Bacteria  which  in  the  past  have  been  isolated  from 
nerve  substance  and  spinal  fluid  in  cases  of  poliomyelitis  can  of  course 
be  excluded  from  etiological  significance  by  the  recent  determination  of 
the  filtrability  of  the  virus  as  determined  by  Flexner  and  Lewis,  and 
Landsteiner  and  Levaditi.  Small  coccoid  forms  in  smears  from  the 
nerve  tissue  recently  described  by  Proescher9  are  of  very  uncertain 
significance.  The  streptococci  recently  described  by  Kosenow  are, 
most  probably,  secondary  invaders.  The  most  important  contribution 
which  has  been  made  in  the  solution  of  this  problem  is  that  of  Flexner 
and  Noguchi.10  These  investigators  placed  small  bits  and  emulsions  of 
the  brain  of  monkeys,  dead  of  poliomyelitis,  in  high  tubes  containing 
human  ascitic  fluid  together  with  a  piece  of  fresh  sterile  rabbit  kidney. 
In  all  essentials  the  method  was  that  followed  by  Noguchi  in  his  culti- 
vation of  Treponema  pallidum.  It  was  necessary  to  use  fresh  unheated 
ascitic  fluid.  Heat  sterilization  rendered  it  unsuitable. 

By  this  method,  after  five  days  opalescence  appeared  about  the 


'Proescher,  N.  Y.  Med.  Jour.,  1913. 

10  Flexner  and  Noguchi,  Jour,  of  Exp.  Med.,  xviii,  1913. 


910  DISEASES  CAUSED  BY   FILTRABLE  VIRUS 

pieces  of  tissue.  This  increased  until  the  tenth  day,  when  sedimenta- 
tion began.  Microscopical  examination  by  Giemsa's  method  of  stain- 
ing revealed  small  globoid  bodies  measuring  from  0.15  to  0.3  micron  in 
diameter,  arranged  in  pairs,  short  chains,  and  masses.  Similar  bodies 
could  later  be  found  in  poliomyelitis  tissues.  Cultures  were  obtained 
from  glycerinated  as  well  as  from  fresh  virus  and  from  the  filtered  as 
well  as  the  unfiltered  material.  Typical  lesions  and  death  have  been 
produced  in  monkeys  with  such  cultures  in  a  few  cases. 

We  have  few  data  which  throw  light  upon  possible  immunity  to  the 
disease.  Repeated  attacks  of  the  disease  in  the  same  human  being 
have  not  been  noted;  but  this,  as  Flexner  and  Lewis  point  out,  may 
be  due  to  the  fact  that  the  epidemics  are  rare,  and  individuals  once 
afflicted  have  passed  beyond  the  susceptible  age  by  the  time  of  the  sec- 
ond epidemic.  As  a  matter  of  fact,  however,  these  workers  have  not 
succeeded  in  reinfecting  monkeys  that  had  recovered. 

In  chickens  a  disease  has  been  observed  similar  in  many  ways  to 
poliomyelitis,  but  further  study  has  shown  this  to  be  a  polyneuritis. 

Of  other  animals  besides  monkeys,  rabbits  only  have  been  success- 
fully inoculated  with  this  disease.  Transmission  to  these  animals  was 
first  reported  by  Kraus  and  Meinicke  11  and  later  by  Lentz  and  Hunte- 
muller. 12  Marks  13  has  studied  the  disease  in  rabbits  thoroughly,  and 
concludes  that  there  is  no  doubt  that  the  virus  can  be  cultivated 
through  a  limited  number  of  generations  in  rabbits.  He  was  able  to 
transmit  to  monkeys  from  rabbit  material.  The  disease,  however,  does 
not  resemble  that  of  man  or  monkeys  clinically  and  no  definite  lesions 
of  the  central  nervous  system  are  present.  The  rabbits  seem  perfectly 
well  for  six  or  seven  days,  when  rapid  weakness  and  death  in  con- 
vulsions occur. 

Animals  which  have  been  unsuccessfully  injected,  even  with  living 
virus,  do  not  develop  immunity.  However,  animals  that  have  been 
successfully  inoculated  and  recovered  are,  like  human  beings,  thereafter 
immune.  Levaditi  and  Landsteiner,  Roemer  and  Joseph,  and  Flexner 
and  Lewis  have  shown  that  the  serum  of  recovered  monkeys  will  pro- 
tect normal  animals  from  fatal  doses  of  the  virus.  That  the  same  pro- 
tective power  for  monkeys  has  been  shown  in  the  serum  of  human 
recovered  cases,  is  shown  by  the  same  authors  and  by  Anderson  and 
Frost  and  consequently  the  intraspinous  injection  of  the  serum  of 

11  Kraus  and  Meinicke,  Deut.  med.  Woch.,  xxxv,  1909. 

12  Lentz  und  Huntemiiller,  Zeitschr.  f.  Hyg.,  Ixvi,  1910. 
"Marks,  Jour,  of  Exp.  Med.,  xiv,  1911. 


ACUTE  ANTERIOR  POLIOMYELITIS  917 

recently  recovered  children  into  patients  in  early  stages  of  the  dis- 
ease has  recently  been  advocated  and  is  though  well  of  by  a  number 
of  observers.  This  work,  however,  has  not  reached  completion  and 
final  judgment  must  be  withheld. 

Flexner  and  Amoss  have  paid  particular  attention  to  the  problem 
of  passive  immunization  and  found  that  in  protecting  monkeys  if  the 
quantity  of  virus  injected  into  the  brain  is  not  too  great  paralysis  can 
be  prevented  in  some  cases  and  delayed  in  others,  by  injecting  the 
serum  of  recovered  monkeys  into  the  subarachnoid  space  by  lumbar 
puncture.  Immunizing  sera  cannot  be  produced  by  treatment  of  in- 
susceptible animals  with  virus,  but  Flexner  and  his  associates  have 
occasionally  succeeded  in  immunizing  actively  by  injecting,  sub- 
cutaneously,  graded  doses  of  crude  virus.  This  method,  however,  is 
not  very  useful,  nor  is  it  very  safe  since  some  of  the  animals 
so  treated  do  not  develop  a  strong  immunity  and  others  may 
become  paralyzed.  It  appears,  therefore,  that  the  neutralizing 
principle,  whatever  it  may  be,  is  present  only  in  animals  and  man  that 
have  recovered  from  actual  infection,  and  the  only  method  of  passive 
immunization  or  serum  treatment,  therefore,  available  at  the  present 
time  is  that  in  which  the  blood  serum  of  individuals  who  have  re- 
covered from  an  attack  is  used. 

Epidemiological  Facts. — To  summarize  the  important  epidemio- 
logical  facts  in  poliomyelitis  we  may  say  that  the  work  of  Flexner 
and  his  group,  as  well  as  that  of  European  workers,  has  shown  that 
the  poliomyelitis  virus  is  present  in  the  mucous  membranes  of  the 
nose  and  throat,  in  the  excretions  from  these  membranes  and  in  the 
intestinal  contents.  It  may  also  be  present  in  the  tonsils.  It  leaves 
the  infected  body  with  the  discharges  from  the  nose  and  throat  and  the 
intestines,  and  when  swallowed  from  the  throat  can  pass  into  the 
intestines,  resisting  the  action  of  the  gastric  and  intestinal  secretions. 
Flexner,  Clarke  and  Dochez  14  have  injected  monkeys  with  filtrates 
from  washings  of  the  intestines  after  feeding  monkeys  with  spinal 
cord  material  from  infected  monkeys  and  taking  the  intestinal  fluid 
two  hours  after  feeding.  Outside  the  human  body  the  virus  probably 
can  survive  for  some  time,  though  the  exact  period  is  not  known. 
Neustaedter  and  Thro  15  claim  to  have  been  successful  in  infecting 
monkeys  with  dust  taken  from  a  sick  room. 


14  Flexner,  Clarke  and  Dochez,  Journ.  A.  M.  A.,  vol.  59,   1912. 

15  Neustaedter  and  Thro,  N.  Y.  Med.  Journal,  94,  1911,  p.  613,  813. 


P18  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

There  is  a  great  deal  of  evidence  to  show  that  poliomyelitis  car- 
riers exist.  Experimental  evidence  of  this  carrier  state  has  been  ad- 
vanced by  Osgood  and  Lucas  16  who  found  the  virus  in  monkeys  five 
months  after  convalescence.  We  ourselves  have  seen  cases  in  which 
it  could  be  definitely  proven  that  they  had  not  been  in  contact  with 
a  preceding  case,  two  of  them  in  country  districts  where  the  scantily 
populated  surrounding  area  could  be  searched  without  danger  of 
overlooking  a  case.  It  is  also  likely  that  at  times  of  epidemic  a 
great  many  very  mild  attacks  of  poliomyelitis  may  occur,  which  are 
mistaken  for  mild  influenza  or  severe  colds,  and  in  connection  with 
the  occurrence  of  a  recent  case  seen  by  us  there  were  a  number 
of  indefinitely  diagnosed  cases  of  intestinal  disturbance,  with  fever, 
in  the  neighborhood.  It  is  not  impossible  that  such  cases  may  be 
true  poliomyelitis  of  a  mild  type  without  paralysis  but  capable  of 
passing  on  the  virus.  Peabody,  Dochez  and  Draper17  have  cited 
similar  cases. 

The  virus  probably  gets  into  the  new  patient  by  direct  and  in- 
direct contact,  and  can  be  carried  from  place  to  place,  perhaps  on  the 
feet  of  flies,  a  fact  which  would  be  indicated  by  some  experiments 
done  by  Flexner. 

In  1911  epidemiologists  of  the  State  Public  Health  Service  of 
Massachusetts  established  a  parallelism  between  the  distribution  of 
poliomyelitis  cases  and  the  occurrence  of  the  biting  stable  fly,  Stomoxys. 
Subsequently,  M.  J.  Rosenau  published  experiments  in  which  he 
obtained  poliomyelitis  infection  by  allowing  infected  Stomxys  flies  to 
bite  monkeys.  These  observations  were  confirmed  by  Anderson  and 
Frost,18  but  all  subsequent  attempts  to  repeat  the  experiments  have 
failed.  The  ordinary  manner  of  infecting  of  the  human  being  is 
probably,  then,  through  the  nasopharynx.  A  great  many  cases  begin 
with  intestinal  disturbances  which  may  last  a  few  days  before  the 
patient  is  ill  enough  to  go  to  bed.  It  is  more  than  likely,  therefore, 
that  the  virus  may  also  enter  the  body  by  ingestion  and  that  infection 
of  food  may  play  a  role. 

The  disease  is  usually  present  to  some  extent  in  crowded  centers 
of  the  world,  in  the  spring  and  summer  months.  The  season  of  greatest 
prevalence  is  May  to  November.  Most  cases  are  in  children  below  five, 
but  adult  cases  do  occur. 


14  Osgood  and  Lucas,  Journal   A.  M.  A.,   57,' 1911,  p.  495. 

17  Peabody,  Draper  and  Dochez,  Monog.  Rock.  Inst.,  No.  4,  Jan.,  1912. 

18  Anderson  and  Frost,  Journ.  A.  M.  A.,  56,  1911,  p.  663. 


ACUTE  ANTERIOR   POLIOMYELITIS  919 

Although  a  great  many  studies  have  been  made  to  trace  the  infec- 
tion of  one  case  to  exposure  to  another,  such  attempts  have  failed  in 
most  instances,  and  it  seems  fairly  well  established  that  there  is  great 
variation  in  the  susceptibility  of  individuals  to  the  disease.  Whether 
this  depends  upon  previous  mild  attacks  of  the  variety  spoken  of 
above  or  whether  it  is  a  congenital  difference,  cannot  be  stated. 


ENCEPHALITIS  LETHARGICA 

It  is  difficult  to  say  whether  the  disease  which  we  now  speak  of 
as  Lethargic  Encephalitis  is  identical  with  the  conditions  formerly 
described  as  "sleeping  sickness,"  "Schlaf  Krankheit,"  etc.  Camera- 
rius,  whom  we  quote  from  Smith,19  is  said  to  have  described  an  epi- 
demic disease  which  occurred  in  Germany  in  1712  which  probably 
represents  the  same  condition.  In  1768  and  in.  1835  similar  epidemics 
seem  to  have  occurred  in  the  trail  of  influenza  outbreaks,  a  fact  which 
is  of  considerable  importance  in  view  of  the  fact  that  recent  interest- 
in  the  disease  dates  from  the  occurrence  of  many  cases  of  lethargic 
encephalitis  which  followed  in  the  train  of  the  last  influenza  epidemic. 
After  the  epidemic  of  1889,  relatively  few  typical  cases  of  what  we 
now  speak  of  as  lethargic  encephalitis  were  reported,  though  nervous 
complications  were  apparently  very  common.  During  the  later  stages 
of  the  great  war  epidemic  of  influenza,  cases  began  to  appear  in  many 
different  places  which,  at  first,  were  either  mistaken  for  poliomyelitis 
or  undiagnosed  before  death.  We  remember  ourselves  seeing  two  cases 
in  soldiers  during  this  period  in  which  diagnosis  was  doubtful  and 
which  we  now  believe  to  have  been  lethargic  encephalitis. 

One  of  the  first  systematic  reports  is  that  of  Economo  20  who  de- 
scribed an  outbreak  of  the  disease  in  Vienna  in  1917.  In  1918  an 
outbreak  occurred  in  Great  Britain,  which  was  studied  and  reported 
by  Wilson,21  Hall,22  Herringham,  and  others.  (The  onset  of  the 
disease  in  America  was  dealt  with  in  an  editorial  in  the  Journal  of 
the  American  Medical  Association,  72,  1919,  414.)  In  speaking  of  the 
distribution  of  the  disease  during  this  last  epidemic,  Smith  states  that 
the  first  cases  occurred  in  Central  Europe  in  1917,  appeared  in  France, 


19  Smith,  U.  S.  Pub.  Health  Eeport,  No.  6,  Vol.  37,  February,  1921. 

20  Economo,  Wien.  klin.  Woch.,  30,  1917,  581. 

21  Wilson,  Lancet,  2,  1918. 

22  Hall,  Brit.  Med.  Jour.,  2,  1918,  467. 


920  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

Great  Britain  and  Algeria  in  late  1917,  and  early  1918,  and  reached 
North  America  during  the  latter  half  of  1918  and  in  early  1919.  The 
disease  spread  rapidly  throughout  the  United  States,  and  Smith  says 
that  by  May,  1919,  cases  had  been  reported  from  twenty  of  the  states. 
The  largest  number  were  reported  from  Illinois,  New  York,  Louisiana, 
and  Tennessee,  a  fact  which  shows  the  apparent  independence  of  the 
disease  of  climatic  conditions.  The  disease  spread  through  the  United 
States  from  east  to  west.  Smith  summarizes  the  epidemiological 
features  as  follows:  In  almost  all  outbreaks  the  appearance  of  epi- 
demic encephalitis  has  been  preceded  by  influenza.  Evidence  of  direct 
communicability  is  lacking,  since  in  analyzing  approximately  900 
people  exposed  in  the  immediate  families  of  cases,  no  secondary  cases 
occurred.  The  age  distribution  is  entirely  different  from  poliomye- 
litis, so  much  so  that  Smith  believes  that  this  alone  would  distinguish 
the  two  diseases.  In  poliomyelitis  over  59  per  cent,  of  the  cases  occur 
before  the  fourth  year,  and  68  per  cent  of  the  cases  occur  below  the 
age  of  5,  whereas  in  epidemic  encephalitis  58  per  cent,  of  the  cases 
occurred  in  individuals  20  years  of  age  and  over.  As  to  sex,  60  per 
cent  of  the  cases  of  encephalitis  occurred  in  males.  Seasonally  the 
apex  of  the  epidemic  curve  in  the  United  States  was  reached  in  March. 

The  relationship  of  the  disease  to  influenza  is  vague,  but  something 
to  be  very  seriously  considered  in  view  of  the  recent  researches  of 
Olitsky  with  influenza  and  of  Strauss  and  Loewe  on  filtrable  virus  in 
encephalitis. 

The  onset  is  usually  gradual,  with  headache,  lassitude  and  grad- 
ually increasing  fever.  Occasionally  there  are  vomiting,  vertigo  and 
muscular  pains.  Disturbances  of  vision  may  appear  early.  Following 
this  there  may  be  an  acute  stage  during  which  the  fever  rises,  vomit- 
ing may  intensify,  there  may  be  disturbances  of  the  cranial  nerves 
and  great  general  weakness  which  gradually  lapses  into  coma.  Occa- 
sionally a  case  may  get  well  in  two  or  three  weeks  following  this,  again 
the  coma  may  persist  for  a  long  time.  Paralysis  of  muscular  groups 
and  facial  paralysis,  ptosis,  disturbances  of  pupil  reflexes  and  other 
muscular  reflexes  occur. 

The  spinal  fluid  shows  an  increased  number  of  cells  in  a  certain 
percentages  of  cases.  The  leucocytes  may  be  slightly  increased,  but 
are  usually  not  very  high.  The  mortality  in  Smith 's  study  was  29  per 
cent. 

The  etiology  of  the  disease  has  been  very  carefully  worked  upon 
since  the  last  appearance  of  the  disease.  A  number  of  bacteria  have 


ACUTE  ANTERIOR  POLIOMYELITIS  921 

been  described  and  isolated  from  cases,  but  very  probably  have  no 
significance.  We  may  dismiss  the  claims  of  bacterial  causative  agents 
as  not  in  any  case  sufficiently  based  on  reliable  evidence.  Of  im- 
portance is  a  publication  by  Strauss,  Hirschfeld  and  Loewe  in  1919. 23 

These  workers  obtained  naso-pharyngeal  mucus  of  fatal  cases  of 
the  disease,  filtered  it  through  Berkefeld  candles  and  injected  it  sub- 
durally  and  intracranially  into  monkeys  and  rabbits.  In  these  animals 
they  produced  the  disease.  A  monkey  (Macacus  Cynomolgus)  in- 
jected on  April  25th  developed  by  May  2d,  lethargy,  general  malaise, 
temperature  and  ptosis  of  the  left  lid,  but  recovered.  Similar  results 
were  obtained  with  a  Rhesus.  Rabbits  intracranially  injected  died  in 
4  or  5  days  with  punctate  hemorrhages  in  the  brain,  intense  conges- 
tion, marked  meningitis  and  mononuclear  infiltrations  about  the  ves- 
sels. They  claim  to  have  repeated  these  experiments  many  times  since 
their  first  publication.  In  1920  Levaditi  and  Harvier  24  claimed  that 
they  confirmed  the  experiments  of  Strauss,  Hirschfeld  and  Loewe, 
both  in  monkeys,  and  in  addition  assert  the  susceptibility  of  guinea 
pigs.  In  a  later  publication  of  Loewe  and  Strauss25  they  state  that 
the  lesions  produced  in  the  brains  of  such  experimental  animals  are 
similar  to  those  seen  in  human  cases,  that  is,  showing  mononuclear 
perivascular  infiltration,  small  hemorrhages  and  general  congestion, 
and  they  add  experiments  in  which  they  succeeded  in  transferring  the 
disease  with  spinal  fluid  and  blood,  as  well  as  with  material  preserved 
in  50  per  cent  glycerine  for  many  months. 

By  means  of  the  Noguchi  anaerobic  tissue-acetic-fluid  method,  they 
report  the  cultivation  of  minute  filtrable  coccoid  bodies,  virtually  iden- 
tical with  the  globoid  bodies  described  by  Noguchi  for  poliomyelitis. 

It  is  not  possible  at  the  present  time  to  make  definite  statements 
concerning  the  reliability  of  these  claims.  Other  observers  of  great 
experience  with  a  large  amount  of  material  have  failed  to  obtain 
similar  results.  The  confirmation  by  Levaditi  and  more  recently  by 
Inmann,  of  Strauss  and  Loewe 's  experiments,  however,  would  en- 
courage the  hope  that  they  are  right.  Moreover,  the  similarity  of  the 
disease  to  poliomyelitis  and  the  general  similarity  of  pathological 
lesions  would  incline  one  to  assume  the  disease  to  be  probably  due  to  a 
filtrable  virus. 


"Strauss,  Hirschfeld  and  Loewe,  N.  Y.  Med.  Jour.,   1919,   772;   Jour.   Infec. 
Dis.,  25,  1919,  378. 

2<  Levaditi  and  Earvicr,  Compt.  Rend,  de  la  Soc.  Biol.,  83,  1920,  354. 
26  Loewe  and  Strauss,  Jour.  Infec.  Dis.,  27,  1920,  250. 


CHAPTER  XL VII 

MEASLES,    SCARLET    FEVER,    MUMPS,    DENGUE    FEVER,    FOOT    AND 

MOUTH  DISEASE 

MEASLES 

THE  causative  agent  of  measles  is  unknown  to  the  present  day, 
and  it  would  be  a  thankless  task  to  review  the  literature  of  the  many 
attempts  to  isolate  microorganisms  from  this  disease,  none  of  which 
has  resulted  in  throwing  any  light  on  the  etiology. 

Attempts  to  produce  the  disease  experimentally  have  frequently 
been  made,  the  earliest  recorded  being  those  of  Home  of  Edinburgh, 
published  in  1759. 1  Home  took  blood  from  the  arms  of  patients  afflicted 
with  measles,  caught  it  upon  cotton,  and  inoculated  normal  indi- 
viduals by  placing  this  blood-stained  cotton  on  wounds  made  in  the 
arm.  Home  claimed  that  in  this  way  he  produces  measles  of  a  modified 
and  milder  type  in  fifteen  individuals.  Home's  results,  however,  while 
at  first  accepted,  were  assailed  by  many  writers  and  it  is  by  no  means 
certain  that  the  disease  produced  by  him  was  really  measles. 

A  number  of  other  observers  after  Home  attempted  experimental 
inoculation  of  this  disease,  and  positive  results  were  reported  by 
Stewart  of  Rhode  Island  (1799),  Speranza  of  Mantua  (1822),  Katowa 
of  Hungary  (1842),  and  McGirr  of  Chicago  (1850). 

The  experiments  of  all  these  early  writers,  however,  are  unsatis- 
factory, owing  to  the  necessarily  unreliable  technique  of  their  methods. 

In  1905  Hektoen2  succeeded  in  experimentally  producing  the  dis- 
ease in  two  medical  students  by  subcutaneous  injection  of  blood  taken 
from  measles  patients  at  the  height  of  the  disease  (fourth  day).  The 
experiments  were  carefully  carried  out  and  the  symptoms  in  the  sub- 
jects were  unquestionable.  They  demonstrated  that  the  virus  of 
the  disease  is  present  in  the  blood.  Attempts  at  cultivation 
carried  out  with  the  same  blood  were  entirely  negative.  It  was  also 


1  Home,  "Medical  Facts  and  Experiments,"  Edinburgh,   1759. 

2  Hektoen,  Jour.  Inf.  Dis.,  ii,   1905. 

922 


MEASLES,  SCARLET  FEVER,  MUMPS,  DENGUE  FEVER,  ETC.      923 

shown  by  Hektoen's  experiments  that  the  virus  of  measles  may  be  kept 
alive  for  at  least  twenty-four  hours  when  mixed  with  ascitic  broth. 

Similar  experiments  were  recently  carried  out  by  Sellards,  both 
on  monkeys  and  on  eight  volunteers,  but  entirely  without  success. 

More  important  than  the  blood  transfer  experiments  from  the 
point  of  view  of  transmission  are  experiments  in  which  inoculation 
has  been  attempted  with  secretions  from  the  nose  and  throat.  In  1852 
Mayer  reported  the  successful  inoculation  of  human  beings  with  mucus 
from  the  noses  and  throats  of  early  measles  cases,  but  complete  failure 
in  similar  attempts  at  transfer  with  skin  desquamations  following  the 
rash.  Anderson  and  Goldberger3  claimed  in  1911  that  they  were  able  to 
produce  temperature  reactions  and  mild  skin  changes  in  monkeys  by 
the  injection  of  nasal  and  pharyngeal  secretions  from  early  cases.  This 
work  has  been  recently  elaborated  and  brought  to  more  convincing 
conclusions  by  Blake  and  Trask.4  These  writers  inoculated  monkeys 
(Macacus  Rhesus)  intratracheally  with  filtered  and  unfiltered  wash- 
ings from  patients  in  the  early  eruptive  stages  of  measles  and  showed 
that  the  lesion  which  developed  in  the  skin  and  buccal  mucous  mem- 
brane during  the  course  of  the  monkey  infection  was  histologically 
almost  identical  with  that  found  in  human  measles.  They  successfully 
transmitted  the  infection  from  monkey  to  monkey  and  demonstrated 
that  one  attack  of  experimental  measles  conferred  immunity  upon  the 
monkeys. 

Epidemiology  and  Prevention. — There  are  few  infectious  diseases 
as  common  as  measles.  Crum  5  has  collected  statistics  which  show 
that  measles  is  responsible  for  about  1  per  cent,  of  all  deaths  occurring 
in  the  temperate  zones.  In  statistical  summaries  of  22  countries  ex- 
tending over  a  period  of  four  years  preceding  1910,  there  were  over 
366,000  deaths  attributable  to  measles  of  an  aggregate  population  of 
32,625,651.  All  races  and  ages  seem  to  be  susceptible,  though  children 
are  more  often  infected,  and  the  discrepancy  between  adults  and  chil- 
dren is  probably  due  merely  to  the  fact  that  most  adults  have  had  the 
disease  before  they  attain  adult  life.  Whenever  young  adults  from 
rural  districts  come  together  in  camps,  epidemics  will  occur  quite  com- 
parable and  more  severe  than  those  occurring  among  school  children 
and  asylum  children  at  an  earlier  period  of  life.  The  disease  is  com- 


8  Anderson  and  Goldberger,  Jour.  A.  M.  A.,  57,  1911,  1612. 

4  Blake  and  Trask,  Jour.  Exper.  Med.,  33,  1921,  385,  413  and  621. 

5  ('mm,  Ainer.  Jour.  Pub.  Health,  4,  1914,  289. 


924 


DISEASES  CAUSED  BY  FILTRABLE  VIRUS 


rnon  all  over  the  world  and  not  apparently  influenced  by  climatic 
conditions. 

When  it  appears  first  among  aboriginal  populations,  it  sweeps 
through  them  with  a  violence  unknown  among  more  civilized  nations 
with  whom  the  disease  has  been  endemic  ,for  centuries.  Such  was 
the  great  epidemic  in  the  Fiji  Islands  in  1874,  and  similar  epidemics 
have  occurred  in  the  South  Sea  Islands  and  among  American  Indians 
and  the  negro  races.  The  disease  occurs  more  commonly  in  cities  than 
in  rural  districts. 

Susceptibility  of  previously  uninfected  individuals  seems  to  be 
practically  universal.  Interesting  in  this  connection  are  the  statistics 
of  concentration  camps  in  the  United  States  during  the  recent  war 
such  as  those  of  Vaughan  and  Palmer6  made  at  Camp  Wheeler.  The 
population  of  this  camp,  like  that  of  many  others,  was  made  up  of 
young  men  from  rural  communities,  many  of  whom  had  not  had 
measles  before.  The  sick  rate  week  by  week  which  followed  the 
onset  of  the  epidemic  is  tabulated  by  Vaughan  and  Palmer  as 
follows : 


For  Week  Ending 

Annual  Measles 
Morbidity  Rate 
per  1000 

October       19 

83 

October       26 

428 

November    2 

615 

November    9 

1760 

November  16 

2200 

November  23 

1120 

November  30 

248 

December     7 

240 

December  14 

19 

We  may  assume  that  the  definite  exposure  to  measles  of  an  unin- 
fected human  being  will  almost  invariably  result  in  an  attack. 

Since  the  disease  is  probably  communicated  by  the  secretions  of 
the  nose  and  throat,  reasonable  exposure  may  be  taken  to  imply 
crowding  in  sleeping  quarters,  contact  in  public  vehicles,  places  of 
amusement,  at  meals,  at  play,  in  schools  and  in  the  ordinary  indoor 
association  of  work  and  recreation.  Whether  or  not  the  disease  can 
be  conveyed  indirectly  to  any  degree  is  not  certain,  but  it  is  very 


'•Vaughan  and  Palmer,  Jonr.  Lab.  and  Clin.  Med.,  4,  1919,  647. 


MEASLES,  SCARLET  FEVER,   MUMPS,  DENGUE  FEVER,  ETC.     925 

likely  that  infection  from  secretions  on  toys,  food  or  other  objects  that 
are  put  into  the  mouth  may  take  place,  so  long  as  the  secretion  is  not 
dried.  Judging  from  what  we  know  or  other  filtrable  virus,  more- 
over, the  virus  of  measles  may  offer  considerable  resistance  even  to 
drying. 

One  of  the  most  important  epidemiological  facts  is  the  infectious- 
ness  of  the  secretions  in  the  early  pre-emptive  stages.  According  to 
Levy  of  Richmond,  the  disease  may  be  infectious  as  long  as  4  days 
before  the  rash  appears  and  since  at  this  time  the  patients  are  rarely 
very  sick,  this  is  the  dangerous  period  for  transmission. 

Uncomplicated  measles  in  itself  is  not  a  very  fatal  disease,  but, 
like  influenza,  measles  seems  to  bring  about  a  certain  susceptibility  to 
various  respiratory  infections,  and  measles  epidemics  are  usually  ac- 
companied by  many  fatal  post-measles  pneumonias.  These  pneu- 
monias may  take  the  form  of  pneumococcus  or  streptococcus  infec- 
tions, according  to  the  nature  of  the  most  prevalent  mouth  and  throat 
flora  prevailing  in  the  community.  The  conditions  for  a  fatal  measles 
epidemic,  therefore,  are  fulfilled  when  measles  breaks  out  in  an  indus- 
trial community,  a  camp,  a  school,  etc.,  during  the  cold  weather  when 
coughs  and  colds  prevail  and  when  virulent  pneumococci  and  strep- 
tococci are  plentifully  scattered  about  in  mucus. 

The  prevention  of  measles,  in  crowded  communities  or  groups,  is 
fraught  with  many  difficulties.  However,  with  vigilance  and  adequate 
discipline,  much  can  be  accomplished.  In  schools,  industrial  commu- 
nities and  in  military  units,  the  most  important  procedure  in  our 
opinion  is  constant  inspection  and  early  segregation  of  all  individuals 
with  catarrhal  colds.  In  the  army  it  has  been  the  practice  of  sani- 
tarians, a  practice  which  we  believe  we  have  seen  succeed  to  an  un- 
expected degree,  to  inspect  entire  units  once  a  day  upon  the  appear- 
ance of  a  case  of  measles.  The  entire  unit  is  made  to  pass  an  in- 
specting officer  in  single  file  in  the  morning,  a  few  questions  *as  to 
general  health  are  asked,  the  conjunctive  and  throats  and  the  skin 
of  the  chest  and  arms  are  rapidly  inspected,  and  individuals 
complaining  of  headache,  a  restless  night,  a  cold  or  a  cough,  or  those 
in  whom  the  conjunctivas  are  inflamed,  or  the  nose  secreting,  are 
made  to  step  out  and,  on  these,  temperatures  are  taken.  All  those 
with  a  temperature  of  100°  or  above  are  isolated  and  great  care  is 
taken  to  segregate  catarrhal  cases  from  the  rest  of  the  population. 
This  method  makes  it  possible  to  inspect  a  large  group  in  a  very  short 
time  and  will  accomplish  far  greater  results  than  the  mere  isolation 


DISKA.sKS    CAUSED    liV    FILTKABLK    V1KIJB 

of  individual  suspicious  eases  which  come  to  the  sanitarian  of  their 
own  accord.  Munson  7  has  given  this  method  particular  attention  in 
the  Jinny  with  astonishingly  favorable  results. 

Since  the  incnliation   time  of  the  disease   is  about   two  weeks,  the 
exclusion  of  children  from  school  need  not  exceed  this  period. 


SCARLET  FEVER 

(Scarlatina) 

The  etiology  of  scarlet  fever,  like  that  of  measles,  is  still  obscure. 
Streptococci  have  been  found  with  striking  regularity  in  the  throats 
of  scarlet-fever  patients,  and  a  large  number  of  investigations  have 
seemed  to  furnish  evidence  for  the  etiological  relationship  of  these 
microorganisms  with  the  disease.  According  to  von  I/mgelsheim, 
Crooke  as  early  as  1885  demonstrated  the  presence  of  streptococci  in 
the  cadavers  nf  sea  Net-fever  victims.  Uaginsky  and  Somrnerfeld  *  in 
1900  examined  a  number  of  scarlatina  cases  with  reference  especially 
to  streptocoecus  infection,  and  reported  the  presence  of  streptococci 
in  the  heart's  blood  of  eight  patients  who  had  died  after  a  very  acute 
and  short  illness.  They  expressed  the  belief  that  the  acuteness  of  the 
illness  and  the  rapidity  of  death  in  these  cases  precluded  the  possi- 
bility of  the  streptococci  being  merely  secondary  invaders.  A  large 
number  of  other  observers  have  expressed  similar  opinions,  but  we 
can  not,  as  yet,  justly  conclude  that  streptococci  are  actually  the 
etiological  agents  of  this  disease. 

Class"  in  1S!M»  described  a  diploeoccus  which  he  cultivated  from  a 
large  number  of  scarlat  ina  patients  and  with  which  he  was  able  to  pro- 
duce exanthemata  and  acute  fever  in  pigs.  Subsequent  investigations 
seem  to  show  that  (lass  was  really  working  with  a  streptococcus. 

IVroser,1"  working  in  Kse.herieh's  clinic,  has  recently  reported  the 
very  favorable  influence  upon  the  course  of  scarlet  fever  of  polyvalent 
streptococcus  antisera.  rrhis  is  not  really  very  strong  evidence  in 
favor  of  the  streptococcus  etiology  of  the  disease,  since  then1  is,  of 
COUrse,  no  doubt  that  streptococcus  infection  complicates  the  disease, 


7  Miuixnit,    Military  Sur^roii,  'Id,    1!H7,  (](\(\. 

"  lttifiintil,-ii   :IM.|    SiHiiHicrfrld,    licrl.    klin.    Worli.,    I'.MK). 

11  r/fi.v.s-.    I'liilM.    M...I.   .lour.,    iii,    1S1MI. 

-.   (|iifiti<i|    by    Ksrhrrii-li,    Wictl.    klil 


MEASLES,  SCARLET  FEVER,   MUMPS,   DENGUE   FEVER,  ETC.     927 

and  it  is  to  be  expected  that  antistreptococcus  serum  should,  therefore, 
benefit  the  patient's  condition  by  combating  this  complication. 

Mallory  u  in  1904  published  observations  on  four  scarlatina  cases 
which  suggested  that  possibily  scarlatina  may  be  caused  by  proto/oa.  Ill 
the  skin,  between  the  epithelial  cells,  he  found  small  bodies  which  were 
easily  stained  with  methylene-blue  and  which  because  of  their  arrange- 
ment and  form  he  interpreted  as  parasites  not  very  unlike  the  plas- 
modium  of  malaria.  Subsequent  investigations  of  Field  12  and  others 
have  failed  to  substantiate  Mallory 's  conclusions.  Mallory  and  Med- 
lar 13  subsequently  described  a  diphtheroid  bacillus  which  they  found 
in  the  tonsils,  throat  and  trachea  of  scarlet  fever  cases.  Another  diph- 
theroid bacillus  has  been  isolated  by  Mallory  and  Parker  from  the 
middle  ear  in  a  number  of  scarlet  fever  cases  complicated  with  -otitis 
media.  The  frequency  with  which  these  bacilli  were  found,  and  the 
fact  that  the  organism  found  by  Mallory  and  Parker  produces  highly 
toxic  substances  in  broth,  makes  it  necessary  to  keep  them  in  mind, 
though  Mallory  and  Parker,  themselves,  make  no  etiological  claims. 

In  1911  Landsteiner,  Levaditi  and  Prasek14  claimed  that  they 
successfully  inoculated  chimpanzees  with  scarlet  fever  by  injecting 
blood  from  patients  and  also  by  rubbing  the  throats  of  the  animals 
with  swabs  taken  t'rom  scarlet  fever  throats.  Hektoen  and  Weller  in 
the  same  year  failed  in  similar  experiments  upon  Macacus  Rhesus. 
Cantacuzene  claims  successful  results  on  a  number  of  lower  monkeys 
with  the  blood  and  lymph-node  suspensions  of  scarlet  /ever  cases. 
Draper  and  I  Ian  ford1 '  have  carefully  gone  over  all  these  investiga- 
tions and  attempted  to  confirm  them  but  completely  failed. 

The  question  concerning  the  etiology  of  scarlet  fever  is  at  the 
present  time  entirely  unsettled.  Unquestionably  the  most  interesting 
line  of  thought  at  the  present  time  is  that  connected  with  the  strep- 
tococci. We  have  mentioned,  in  the  streptococcus  chapter,  the  work  of 
Uaginsky  and  Sommerfeld,16  and  there  is  no  question  about  the  fact 
that  a  severe  hemolytic  infection  of  the  throat  is  an  invariable  accom- 
paniment of  scarlet  fever.  In  the  series  studied  by  Baginsky  and 
Sommerfeld  there  were  8  in  which  the  streptococcus  infection  accom- 


11  Mallory,  Jour.  Mod.   Research,  x.    1D04. 

12  Field,  Jour.   KXJHM-.   Mod..   7,   liH>f>. 

13  Mallorii  and  Medlar,  Jour.  Mod.  Res.,  34,  1916. 

14  LtiH(lst< -incr,  Leraditi  and  /V<wA\  Ann.  do  I 'lust.  Past.,  25,  1011,  754. 
i:>  DnttH T  and   llanford,  Journ.   Kxp.   Mod.   Vol.   17.   ItM.'i.  p.  f>17. 

111  />(/</ ///,s'A\(/  and  Sommerfeld. 


928  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

panying  scarlatina  was  so  violent  that  it  could  hardly  be  regarded 
as  a  mere  secondary  infection,  and  the  writer,  like  many  others,  has 
seen  a  number  of  cases  in  which  severe  streptococcus  infection  of  the 
throat,  rapidly  followed  by  general  septicemia,  was  accompanied  by 
a  scarlatiniform  rash,  and  in  such  cases  it  is  impossible  to  say  whether 
one  is  dealing  with  a  fulminating  scarlet  fever  case  or  with  a  violent 
streptococcus  infection  with  a  toxic  rash.  The  experiments  of  Tuni- 
cliff u  seemed  to  show  definitely  that  scarlet  fever  blood  contains 
specific  increase  of  opsonins  for  hemolytic  streptococci  and  that  strep- 
tococci from  scarlet  fever  cases  fall  into  a  homologous  group  by  ag- 
glutination reactions.  Moser  and  Von  Pirquet18  in  1903  had  claimed 
that  the  blood  of  scarlet  fever  patients  quite  frequently  agglutinated 
streptococci  and  also  stated  that  the  streptococci  isolated  from  scarlet 
fever  blood  could  be  agglutinated  specifically  in  most  cases  with 
immune  sera  produced  with  such  streptococci,  while  streptococci 
isolated  from  other  sources  were  rarely  so  agglutinated.  This  ap- 
parent specificity  of  the  scarlet  fever  streptococci  has  recently  been 
more  carefully  investigated  by  Bliss*  in  Dochez's  laboratory,  and 
in  spite  of  the  technical  difficulties  of  agglutination  experiments  with 
this  group,  these  workers  too  seemed  to  have  found  a  certain  amount 
of  specificity  in  the  scarlet  fever  streptococcus  group.  In  view  of 
the  toxic  substances  which  Zinsser,  Parker  and  Kuttner19  have  recently 
obtained  from  streptococcus  cultures,  toxic  substances  which  are  not 
regarded  as  specific  exotoxins  and  are,  therefore,  not  to  be  confused 
with  previous  claims  of  exotoxin  production  by  streptococci,  and  in 
consideration  of  the  nature  of  the  scarlet  fever  rash,  we  do  not  believe 
it  impossible  that  the  skin  manifestations  of  scarlet  fever  may  be  toxic 
in  nature,  and  that,  while  many  features  in  the  disease  would  suggest 
a  filtrable  virus  similar  to  that  of  measles,  there  is  still  much  logic 
in  continuing  bacteriological  investigations. 

Epidemiology  and  Prevention. — There  seems  to  be  no  doubt  about 
the  fact  that  the  disease  is  transmitted  by  the  nasal  and  pharyngeal 
mucus  at  all  stages  of  the  disease.  The  disease  is  probably  com- 
municable from  the  very  beginning  of  the  onset  of  symptoms  in 


17  Tunicliff,  Jour.  Infec.  Dis.,  Journ.  A.  M.  A.,   1920,   74,  p.   1386  and  75,  p. 
1339;  also  Journ.  Inf.  Dis.,  29,  p.  91,  1921. 

18  Moser  and  Von  Pirquet,  Cent.  f.  Bakt.,  Orig.,  34,  1903,  560  and  714. 
*  Bliss,  Bull.  Johns  Hopk.  Hosp.,  31,  1.920,  p.  173. 

19  Zinsser,   Parker   and   Kuttner,   Proc.    Soc.    for   Exper.    Biol.    and   Med.,    18, 
1920,  49. 


MEASLES,  SCARLET  FEVER,   MUMPS,  DENGUE  FEVER,  ETC.      929 

the  throat.  It  remains  contagious  throughout  the  disease  and  far 
into  the  convalescent  period.  In  ordinary  uncomplicated  cases  the 
infectiousness  probably  ends  three  or  four  weeks  after  disappearance 
of  the  rash,  but  when  suppurating  ears  or  other  open  secondary 
lesions  persist,  contagiousness  may  last  throughout  the  period  of 
the  existence  of  the  secondary  lesion.  Place20  has  particularly 
studied  such  cases,  and  reports  isolated  observations  in  which  con- 
tagiousness has  lasted  for  twenty  weeks  after  convalescence. 

The  so-called  "return"  cases  are  due  to  this  long  period  of 
contagiousness,  and  Rosenau  states  that  in  the  Boston  City  Hospital 
it  has  been  observed  that  about  1.5  per  cent  of  discharged  scarlatina 
convalescents  give  rise  to  " return"  cases,  although  the  patients  are 
kept  in  the  Hospital  for  fifty  days.  From  our  own  observations 
in  army  sanitation,  there  is  very  little  doubt  in  our  minds  about 
the  existence  of  scarlet  fever  carriers  and  this  is  in  keeping  with 
the  observations  of  others. 

We  have,  thus,  as  dangers  for  scarlet  fever  transmission,  the 
typical  cases  themselves  from  the  beginning  of  the  throat  infection 
until  long  after  convalescence,  the  cases  in  which  persistent 
secondary  suppurative  lesions  continue  after  convalescence,  the  mild 
unrecognized  cases  and,  possibly,  carriers. 

In  addition  to  this,  transmission  by  milk  has  not  been  uncommon. 
Trask21  has  collected  thirty-five  scarlet  fever  epidemics  indirectly 
traceable  to  milk,  and  Rosenau  speaks  of  a  milk  epidemic  in  Boston 
which  gave  rise  to  500  cases.  This  epidemic  was  suppressed  by 
pasteurization  of  the  milk. 

In  prevention  of  the  disease  attention  must,  therefore,  be  chiefly 
centered  upon  early  recognition  and  proper  quarantine.  In  schools, 
asylums  and  other  closely  associated  units,  great  care  must  be  exer- 
cised to  detect  the  first  symptoms  of  sore  throat  when  scarlet  fever 
has  occurred  in  any  member  of  the  community,  and  daily  inspection 
is  as  necessary  here  as  in  measles.  Children  from  households  in 
which  scarlet  fever  has  occurred  should  be  excluded  from  school 
and  isolated  until  the  incubation  period  of  seven  days  is  over.  The 
incubation  in  this  disease  is  usually  shorter  than  this,  rarely  longer. 
The  isolation  of  cases  should  be  continued  for  from  fifty  to  sixty 


20  Place,  cited  from  Rosenau 's  Preventive  Medicine  and  Hygiene,  D.  Appleton 
&  Co.,  New  York  and  London,  1921. 

21  Trask,  U.  S.  Pub.  Health  Bulletin,  No.  41. 


930  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

days,  and  no  case  should  be  returned  to  ordinary  life  as  long  as 
middle  ear  disease  or  any  other  open  suppurative  lesion  persists. 


MUMPS 

Mumps  is  one  of  the  most  difficult  infections  to  circumscribe 
when  once  it  has  started  in  a  crowded  community.  Mumps  epidemics 
in  the  army  spread  with  speed  and  without  yielding  to  ordinary 
preventive  measures.  Ordinarily  it  is  chiefly  a  disease  of  children 
among  whom  it  spreads  in  schools  and  institutions. 

Prevention  is  particularly  difficult  because  the  susceptibility 
among  children  is  practically  universal  and  since  exposure  need  not 
be  very  close  to  give  rise  to  infection.  Also,  difficulties  are  added 
to  by  the  fact  that  it  may  be  passed  on  to  others  during  the  incuba- 
tion time  before  actual  symptoms  have  appeared.  Our  impression 
from  army  experience  is  that  there,  may  be  carriers. 

Infection  is  direct,  by  the  secretions  of  the  mouth,  nose  and 
throat,  and  it  enters  the  new  victim  probably  by  the  same  route. 
The  incubation  time  after  infection  may  be  anywhere  from  five 
days  to  three  weeks.  Martha  Wollstein22  has  brought  forward 
evidence  which  indicates  that  the  saliva  and  secretions  from  mumps 
cases  contain  a  filtrable  virus.  With  such  filtered  secretions  she 
obtained  pathological  changes  in  the  testicles  and  parotid  glands  of 
cats  which  simulated  human  mumps.  The  most  serious  complications 
are  those  occurring  in  the  testes  which  in  male  adults  may  have 
serious  consequences. 

One  attack  usually  protects,  though  not  always. 

Prevention  depends  upon  early  recognition  and  isolation  for  two 
weeks  after  all  symptoms  have  disappeared. 

When  the  disease  spreads  in  a  group,  it  should  be  remembered 
that  it  may  be  infectious  for  some  time  before  symptoms  occur  and 
that  the  incubation  time  may  last  as  long  as  three  weeks.  Protective 
measures,  exclusion  of  contacts  from  school,  and  closure  of  schools 
if  found  necessary,  must  cover  this  period. 


22  Wollstein,  M.,  Jour.  Exper.  Med.,  23,  1916,  265. 


MEASLES,  SCARLET  FEVER,  MUMPS,  DENGUE  FEVER,  ETC.     931 


DENGUE  FEVER 

Dengue  fever  is  of  sanitary  importance  because  it  spreads  rapidly, 
occurs  epidemically  and  may  lead  to  a  very  high  sick  rate,  though 
it  rarely  kills.  It  is  tropical  in  its  distribution,  but  has  occurred 
epidemically  in  the  sub-tropical  countries.  Southern  and  Eastern 
Europe  have  had  small  epidemics  and  an  occasional  sporadic  dis- 
tribution of  cases. 

The  onset  is  sudden  and  begins  with  severe  pains  throughout  the 
body,  weakness  and  chilliness.  There  is  conjunctival  injection  and 
there  may  be  gastro-intestinal  symptoms.  The  temperature  rises  to 
103°  or  104°  and  with  this  there  is  headache  and  the  pains  in  the 
back  and  legs  increase.  The  pain  seems  to  be  largely  localized  in 
the  muscles.  The  fever  persists  usually  for  about  three  days  when 
it  drops  considerably,  and  remains  down  for  two  or  three  days  when 
it  rises  again.  Usually  at  this  time  there  is  a  rash  on  the  hands 
which  spreads  to  the  arms,  trunk  and  legs.  Castellani  and  Chalmers23 
describe  this  as  a  "measly"  eruption  in  most  cases,  though  in  others 
it  may  resemble  that  of  scarlet  fever.  After  the  second  febrile  attack, 
convalescence  is  usually  rapid.  The  mortality  of  the  disease  is 
negligible. 

In  1903  Graham,24  working  in  Syria,  made  blood  examinations 
on  a  large  number  of  cases  of  Dengue  and  described  protozoa-like 
organisms  within  the  red  blood  cells.  He  believed  that  the  disease 
was  transmitted  by  the  ordinary  mosquito,  Culex  fatigans.  The 
geographical  distribution  of  this  mosquito  corresponds  fairly  well  with 
that  of  the  disease.  In  one  case  he  produced  the  disease  in  man 
by  the  subcutaneous  injection  of  a  suspension  of  salivary  glands 
of  infected  mosquitoes.  He  produced  the  diseases  by  bites  of  the 
mosquitoes  that  had  fed  on  Dengue  patients.  In  1906  Bancroft 
produced  typical  attacks  in  two  volunteers  by  the  bites  of  Stegomya 
fasciata  which  had  been  allowed  to  feed  on  Dengue  patients  on  the 
second  day  of  the  disease.  Of  considerable  importance  are  the  ex- 
periments of  Ash  burn  and  Craig25  of  the  United  States  Army  which 
were  carried  out  in  the  Philippine  Islands  in  1907.  These  inves- 

M  Castellani  and  Chalmers,  Textbook  of  Tropical  Medicine,  W.  Wood  & 'Co., 
Now  York. 

"(irahatn,  Jour.  Trop.  Mod.,  6,  1903,  209. 

25  Ashburn  and  Craig,  quoted  from  Craig,  Jour.  A.  M.  A.,  75,  1920,  1171. 


932  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

tigators  failed  to  confirm  Graham's  work  regarding  the  presence 
of  protozoa-like  organisms  in  the  blood.  They  produced  the  disease 
in  volunteers  with  unfiltered  blood  of  Dengue  cases.  They  then 
injected  two  volunteers  intravenously  with  diluted  defibrinated 
blood  of  Dengue  patients,  filtered  through  filter  candles  which  held 
back  Micrococcus  melitensis  and  the  cholera  spirillum.  Both  of 
these  volunteers  developed  typical  attacks  in  about  three  days.  They 
subsequently  confirmed  Graham's  assertion  concerning  the  trans- 
mission  of  the  disease  by  Culex  fatigans.  They  also  showed  that 
the  disease  is  not  contagious  in  the  ordinary  sense.  Craig  suggests 
the  possibility  of  the  disease  being  caused  by  a  spirochaete,  basing 
his  opinion  on  certain  analogies  with  yellow  fever  as  brought  out 
by  Nbguchi's  researches.  However, 'there  is  still  considerable  uncer- 
tainty concerning  this. 


FOOT-AND-MOUTH    DISEASE 

This  malady  occurs  chiefly  in  cattle,  sheep,  and  goats,  more  rarely 
in  other  domestic  animals.  It  is  characterized  by  the  appearance 
of  a  vesicular  eruption  localized  upon  the  mucosa  of  the  mouth  and 
upon  the  delicate  skin  between  the  hoofs.  In  the  females  similar 
eruptions  may  appear  upon  the  udders.  With  the  onset  of  the 
eruption  there  may  be  increased  temperature ;  refusal  of  food,  and 
general  depression.  Usually  the  disease  is  mild ;  the  vesicles  become 
small  ulcers  and  pustules.  Occasionally  the  disease  is  complicated 
by  catarrhal  gastroenteritis  or  an  inflammation  of  the  respiratory 
tract,  and  death  may  ensue. 

The  disease  is  transmitted  from  animal  to  animal  by  means  of 
virus  contained  in  the  vesicular  contents.  Infection  may  also  take 
place  through  the  agency  of  milk. 

Rarely  the  disease  may  be  transmitted  to  man.  Such  infection, 
when  it  does  take  place,  occurs  usually  among  the  milkers  and 
attendants  in  dairies,  and  is  transmitted  by  direct  contact.  The 
disease  in  man  is  usually  very  mild.  Mohler  states  that  the  disease 
may  be  transmitted  to  man  by  the  milk  of  infected  animals.  He26 
adds  that  in  the  United  States  the  disease  has  been  practically 
eradicated. 

M  Mohler,  Bull.  No.  41,  U.  S.  Pub.  Health  and  Mar.  Hosp.  Serv.,  Wash.,  1908. 


MEASLES,  SCARLET  FEVER,  MUMPS,  DENGUE  FEVER,  ETC.     933 

The  causative  agent  of  foot-and-mouth  disease  is  unknown.  A 
number  of  organisms  have  been  cultivated  from  the  vesicles  and 
mucous  membranes  of  afflicted  animals,  but  none  of  these  could  be 
shown  to  have  etiological  significance.  Loeffler  and  Frosch,27  have 
demonstrated  that  the  virus  contained  in  the  vesicles  may  pass 
through  the  pores  of  a  filter.  The  virus  is  easily  destroyed  by 
heating  to  60°  C.  and  by  complete  desiccation. 

One  attack  of  foot-and-mouth  disease  protects  against  subsequent 
attacks.  This  immunity  in  most  cases  lasts  for  years,  though  rare 
cases  of  recurrence  within  a  single  year  have  been  reported.  Loeffler 
has  actively  immunized  horses  and  cattle  with  graded  doses  of  virus 
obtained  from  vesicles  and  with  the  sera  of  such  animals  has 
produced  passive  immunity  in  normal  subjects. 

27  Loeffler  und  Frosch,  Cent,  f .  Bakt.,  1,  1908. 


CHAPTER   XLVITI 

TYPHUS  FEVEE,  TEENCH  FEVEK,  KOOKY  MOUNTAIN  SPOTTED 
FEVEE.  NOTES  ON  DELOUSING  AND  A  CONSIDEEATION  OF  THE 
SO-CALLED  EICKETTSIA  BODIES. 

IN  this  chapter  we  have  brought  together  a  group  of  diseases 
which  are  insect  borne  and  in  which  the  etiological  factor  at  the 
present  time  is  uncertain.  We  have  attached  to  the  end  of  the 
chapter  a  short  resume  of  Rickettsia  bodies,  appearances  to  which 
allusion  is  made  in  consideration  of  all  the  diseases  here  dealt  with. 
The  chapter  has  not  been  introduced  with  these  Rickettsia  bodies 
because  we  do  not  feel  at  the  present  time  that  their  relationship 
to  the  diseases  or  even  their  positive  interpretation  as  living  organ- 
isms has  been  fully  established.  The  evidence  which  connects  them 
with  the  diseases  in  question,  however,  is  growing  so  important  that 
general  information  as  to  their  nature  and  appearance  cannot  be 
ignored.  In  reading  the  chapters,  if  the  reader  finds  reference  to 
Rickettsia  bodies  in  the  sections  on  the  diseases,  he  is  referred  for 
further  information  to  the  section  on  Rickettsia  at  the  end  of  the 
chapter. 

TYPHUS   FEVEE 

Typhus  fever  is  an  infectious  disease  which  is  characterized  by 
an  incubation  time  of  five  days  or  more,  high  temperature,  and  a 
petechial  rash.  It  has  been  characterized  as  peculiarly  a  disease 
of  filth  and  has  epidemically  disappeared  in  most  of  the  civilized 
countries,  although  it  is  still  endemic  in  certain  parts  of  Europe, 
North  and  South  America,  and  occurs  epidemically  in  Mexico  under 
the  name  of  Tabardillo.  In  New  York  it  has  recently  been  found 
to  exist  not  infrequently.  It  was  described  as  a  new  clinical  entity 
by  Brill,  and  has  been  spoken,  of  as  Brill 's  disease,  but  the  work  of 
Anderson  and  Goldberger  has  shown  that  Brill's  disease  is  identical 
with  typhus  fever. 

During  the  present  war  great  epidemics  occurred  in  the  countries 
of  Eastern  Europe,  an  epidemic  of  great  destructiveness  sweeping 

934 


TYPHUS   FEVER,   TRENCH   FEVER,   ETC.  935 

over  Serbia  during  the  winter  of  1914  to  1915 ;  and  the  disease 
occurred  in  the  Russian,  Hungarian,  Austrian,  Balkan  and  Turkish 
Armies  throughout  the  subsequent  years.  At  the  present  writing 
there  is  much  typhus  in  Russia,  Serbia,  Poland  and  in  parts  of 
Turkey. 

The  Disease. — Among  clinical  descriptions  of  typhus  the  first 
important  scientific  ones  were  those  of  Gerhard,  Jenner  and  Murchi- 
son.  The  differentiation  between  typhoid  and  typhus  exanthematicus 
was  first  made  by  Gerhard  in  1837.  Since  then,  the  disease  has  been 
clinically  well  characterized  and,  indeed,  is  not  easy  to  mistake  for 
any  other  malady  if  once  observed  in  a  typical  case. 

The  incubation  time  may  range  from  five  to  twenty-one  days. 
A  case  of  autopsy  infection  published  during  the  war  took  exactly 
twelve  days  to  develop. 

The  onset  may  vary  from  extreme  abruptness  to  a  more  gradual 
one.  We  take  the  main  points  of  our  description  from  a  recent  study 
by  George  C.  Shattuck1  on  cases  observed  during  the  Serbian 
epidemic. 

The  temperature  rises  rapidly,  often  to  from  103°  to  104°,  with 
chills,  great  depression,  weakness,  pains  in  the  head  and  limbs.  The 
eruption  appears  on  the  fourth  or  fifth  day  after  the  onset  and, 
except  in  times  of  epidemic,  the  diagnosis  is  extremely  difficult  in 
the  pre-eruptive  stage.  As  the  eruption  appears  the  fever  is  apt 
to  rise.  The  rash  begins  to  appear  usually  on  the  shoulders  and 
trunk,  extending  secondarily  to  the  extremities,  the  backs  of  the 
hands  and  feet,  and  sometimes  to  the  palms  and  soles.  It  becomes 
more  abundant  during  the  subsequent  second  and  third  days,  but 
it  is  seen  very  rarely  on  the  face  and  forehead.  The  rash  is  at  first 
composed  of  pink  spots  which  disappear  on  pressure,  but  soon  it 
becomes  purplish,  more  deeply  brownish-red  and  finally  fades  into 
a  brown  color.  Hemorrhagic  centers  may  later  develop  which  persist 
for  considerable  lengths  of  time.  Shattuck  saw  no  eruptions  on 
the  mucous  membranes  of  the  mouth  and  pharynx.  It  is  important 
to  remember  (a  thing  which  Shattuck  points  out  and  which  we  have 
confirmed)  in  connection  with  the  differential  diagnosis  between 
typhus  and  purpura  haemorrhagica  that  in  the  purpura,  eruption 
the  spots  are  hemorrhagic  from  the  beginning  and  are  more  sharply 


1  Kliattuck,    Typhus    TYvor,    etc.,    Rep.    Amor.    Rod.    Cross,    Sorb.    Epidemic, 
Harvard  Univ.  Press,  1920. 


936  DISEASED  CAUSED  BY  FILTKABLE  VIRUS 

defined  than  those  of  typhus.  Fresh  flea  bites  are  sometimes  hard 
to  distinguish  from  the  typhus  eruption. 

The  heart  is  usually  rapid  and  may  become  irregular.  The  blood 
pressure  is  apt  to  be  low  and  Shattuck  believes  that  myocardial 
weakness  often  occurs.  Epistaxis  may  occur  at  the  height  of  the 
disease.  Bronchitis  often  occurs  during  the  later  stages,  and  cough 
is  almost  regularly  present.  Nervous  symptoms  of  various  kinds 
are  important  accompaniments  of  the  disease.  In  many  cases  a 
state  of  lethargy  resembling  that  of  typhoid  fever  is  present.  There 
may  be  twitching  of  the  muscles  during  this  stage  of  stupor. 
Delirium  occurs  in  severe  cases. 

The  leucocytes,  as  worked  out  by  Sellards,2  are  rarely  increased 
in  number,  ranging  in  number  from  3,000  to  15,000,  the  average 
being  between  5,000  and  7,000.  Differential  counts  show  ap- 
proximately normal  percentages. 

The  most  common  complications  are  parotitis,  suppurative  otitis 
and  mastoiditis,  and  a  peculiar  gangrene  of  the  extremities,  es- 
pecially of  the  feet,  which  is  particularly  associated  with  cases 
occurring  during  the  cold  weather.  This  gangrene  is  characteristic 
of  the  disease  and  is  probably  associated  with  the  vascular  changes 
incident  to  the  localization  of  the  virus.  Bronchitis  is  almost  a 
regular  complication.  Albuminuria  is  present,  and  the  urine  gives 
a  Diazo  reaction. 

For  a  thorough  discussion  of  the  pathology  of  the  disease  we 
refer  the  reader  to  Wolbach's  Harvey  Lecture.  (Series  1920-1921, 
New  York  Harvey  Society.) 

Epidemiology. — Hirsch3  in  his  Handbook  of  Geographical  and 
Historical  Pathology,  associated  typhus  fever  with  the  dark  days 
of  the  history  of  the  world,  war  and  famine.  Typhus  fever  epidemics 
have  probably  decimated  populations  and  armies  far  back  into  the 
history  of  the  Middle  Ages,  and  probably  before.  An  epidemic  of 
what  is  probably  typhus  fever  was  spoken  of  in  the  Chronicles  of 
Joinville  as  almost  destroying  the  Christian  Armies  near  Salonika. 
Great  epidemics  ravaged  Central  and  Eastern  Europe  in  the  Eigh- 
teenth Century,  and  the  disease  was  prevalent  in  England  and 
Ireland  at  this  time.  Epidemics  occurred  in  Northern  England  and 


2  Sellards,   Typhus   Fever,   etc.,   In   the   Serbian    Epidemic,   Red   Cross   Repoit, 
Harvard  Univ.  Press,   1920. 

9  Hirsch,  Indenham  Society  Publication,  London,  1888. 


TYPHUS   FEVER,   TRENCH   FEVER,   ETC.  937 

in  Dublin  in  the  Nineteenth  Century,  and  Strong4  states  that  in 
Dublin  alone,  in  the  epidemic  of  1846,  60,000  people  died  of  the 
disease.  In  Mexico  the  disease  has  been  endemic  since  the  early 
part  of  the  Sixteenth  Century,  and  here,  as  in  South  America,  it 
is  known  as  Tabardillo. 

Hirsch  states  (we  quote  from  Strong),  that  of  147  epidemics 
which  occurred  in  temperate  and  cold  latitudes,  30  reached  their 
heights  in  the  spring,  28  in  the  winter  and  spring,  21  in  the  spring 
and  summer,  and  19  in  the  summer  and  autumn.  As  a  matter  of 
fact,  the  disease  is  one  of  relatively  cold  climates  and  elevated 
plateau  countries.  Nevertheless,  it  is  also  endemic  in  such  places 
as  the  North  of  Africa,  where  Nicolle5  made  his  important  dis- 
coveries. In  some  large  cities  of  countries  that  are  not  ordinarily 
visited  by  typhus  epidemics,  the  disease  has  remained  endemically 
prevalent  among  those  parts  of  the  population  living  under  unclean 
conditions.  This  is  the  case  in  New  York  where  the  disease  has  been 
prevalent  in  a  mild  form  for  a  great  many  years. 

Transmission  is,  as  far  as  we  know  at  the  present  time,  entirely 
by  the  agency  of  lice.  The  idea  that  lice  were  concerned  in  the 
disease  is  not  a  new  one.  We  find  in  Strong's  study  of  the  literature 
that  Murchison  suggested  it  in  1876.  Cortezo  made  a  similar  state- 
ment in  1903,  basing  the  opinion  purely  on  clinical  observation. 
The  matter  was  not  settled  until  1909  when  Nicolle  proved  louse 
transmission  by  infecting  a  chimpanzee  with  typhus  blood  and  sub- 
sequently showing  that  the  disease  could  be  transmitted  to  monkeys 
by  the  bites  of  infected  body  lice,  as  well.  This  important  result 
was  confirmed  in  1911  by  Ricketts  and  Wilder6  in  Mexico,  and  in 
the  same  year  by  Anderson  and  Goldberger7  of  the  United  States 
Public  Health  Service.  Since  that  time  many  confirmatory  observa- 
tions have  been  made. 

Whether  or  not  other  methods  of  transmission  are  possible  is 
still  somewhat  in  doubt.  It  is  of  course  certain  that  direct  trans- 
mission of  blood  from  an  infected  case  can  cause  the  disease,  and 


4  Strong,  Typhus  Fever,  etc.,  Kep.  Red  Cross,  Serbian  Epidemic,  Harv.  Univ. 
Press,  1920. 

5  Nicolle,   Compt.   rend.   Acad.   d.   Sc.    1909,   157,   Ann.   de   I'lnst,   Past.,   1910, 
1911,  1912. 

'Ricketts  and  Wilder,  Jour.  Infec.  Dis.,  July,  1911,  p.  9. 

7  Anderson  and  Goldberger,  Pub.  Health  Rep.,  Washington,  March,   1912  and 
May  31,  1912. 


938  DISKASKS  CAUSED  BY  FILTRABLE   VIRUS 

autopsy  infections  have  been  observed,  but  this  mode  of  transmission 
can,  of  course,  play  no  role  of  importance  in  epidemic  transmission. 
It  was  suspected  for  a  time  that  the  sputum  of  typhus  eases  during 
the  stage  of  bronchitis  might  prove  infectious  and  doctors  and  nurses 
in  Serbia  for  a  time  wore  masks.  But  no  clear  evidence  of  any 
such  accident  has  been  brought  to  our  knowledge,  and  as  far  as 
we  know  at  the  present  time  the  louse  is  the  only  important  means 
by  which  the  disease  is  conveyed.  Indeed,  our  own  experiences  with 
lice  would  persuade  us  that  it  is  very  difficult  to  absolutely  exclude 
the  bite  of  a  louse  in  an  infected  case,  for  lice  may  lodge  on  the 
body  in  spite  of  the  most  rigid  precautions  and  the  bite  of  a  louse 
may  be  entirely  painless  and  without  noticeable  reaction  in  many 
individuals. 

There  has  been  a  considerable  amount  of  discussion  as  to  whether 
the  headlouse  can  transmit  the  disease  as  well  as  the  body  louse. 
Goldberger  and  Anderson  at  one  time  believed  this  to  be  the  case, 
but  there  is  still  much  uncertainty  about  it,  and  the  weight  of 
evidence  seems  to  be  against  it.  As  a  matter  of  fact,  in  individuals 
who  are  sufficiently  lousy,  specimens  of  the  body  louse  variety  may 
be  found  in  the  hair  of  the  head,  neck  and  beard,  on  occasion. 

These  facts  explain  clearly  why  typhus  epidemics  occur  under 
conditions  of  crowding,  poverty  and  war ;  why  they  spread  so  easily 
from  patient  to  doctor,  and  why  they  occur  chiefly  in  cold  countries 
at  times  of  the  year  when  human  beings  are  less  apt  to  bathe  and 
keep  clean  and  more  likely  to  live  close  together  in  crowded  quarters 
for  the  sake  of  warmth.  The  louse,  also,  is  not  a  tropical  insect, 
but  thrives  particularly  in  the  cooler  countries.  In  Mexico,  for 
instance,  as  Ricketts  and  Wilder  found,  there  was  little  or  no  typhus 
fever  in  the  lowlands  near  the  coast  where  lice  were  less  common 
than  fleas,  but  typhus  was  most  prevalent  on  the  cooler  plateau 
country  about  the  City  of  Mexico,  where  the  population  was  much 
more  commonly  infested  with  lice.  • 

The  Serbian  epidemic  of  1915  was  so  severe  that  it  interfered 
materially  with  military  activities,  and  it  was  probably  because  of 
the  epidemic  that  the  Austrian  Armies  delayed  their  second  attack 
upon  Serbia.  A  detailed  description  of  this  epidemic  is  found  in 
the  article  by  Strong  in  the  Red  Cross  Report  referred  to  above. 
Typhus  appeared  in  the  Serbian  Army  in  October  and  November 
of  1914,  and  it  is  said  by  Strong  to  have  been  introduced  from 
Albania.  It  is  also  believed  that  typhus  was  present  in  the  Austrian 


TYPHUS  FEVER,   TRENCH  FEVER,   ETC.  939 

• 

Army  during  its  first  invasion  of  Serbia,  and  there  may  well  have 
been  endemic  cases  in  Serbia  before  the  invasion.  After  the  Aus- 
trian repulse  of  the  late  summer  of  1914,  typhus  broke  out  among 
Austrian  prisoners  and  Serbian  soldiers.  Owing  to  the  fighting  in 
the  north  the  Serbian  civilian  population  was  forced  southward 
and  conditions  of  personal  hygiene,  housing,  etc.,  were  made  par- 
ticularly difficult.  There  was  insufficient  shelter,  and  the  cold 
autumn  weather,  the  lack  of  clothing,  etc.,  brought  about  conditions 
of  crowding  and  a  tendency  to  remain  close  in  quarters  and  wear 
whatever  clothes  the  people  had,  without  changing  for  long  periods 
at  a  time.  Filth  and  lousiness  naturally  resulted,  and  ideal  condi- 
tions for  typhus  dissemination  were  created.  In  addition  to  this,  short- 
age of  food  and  the  hardships  attendant  upon  the  general  conditions 
reduced  resistance.  It  is  natural  that  under  the  circumstances  very 
little  was  done  at  the  beginning  of  the  outbreak  to  circumscribe 
the  disease,  and  it  is  doubtful  whether  this  would  have  been  possible. 
By  January  of  1915  the  epidemic  had  begun  to  spread  throughout 
the  country  from  that  period  on,  and  reached  its  height  in  March 
and  April.  According  to  Strong,  at  the  height  of  the  epidemic 
cases  were  appearing  at  the  rate  of  9,000  a  day.  It  is  estimated 
that  the  mortality  at  the  height  of  the  epidemic  ranged  between 
30  and  60  per  cent,  and  that  150,000  deaths  occurred  within  six 
months. 

Animal  Transmission. — One  of  the  most  important  steps  of  course 
in  etiological  study  of  an  infectious  disease  is  the  production  of  the 
disease  in  animals.  In  the  case  of  typhus  this  was  first  accomplished 
by  Nicolle. 

In  1909  Nicolle8  successfully  inoculated  an  anthropoid  ape,  and 
Anderson  and  Goldberger9  in  the  same  year  succeeded  in  inoculating 
lower  monkeys,  rhesus  and  capuchin.  Similar  successful  monkey 
inoculations  were  made  by  Ricketts  and  Wilder,10  by  Gavino  and 
Girard.11  In  these  animals  inoculation  with  blood  from  active  cases 


8  Nicolle,  Compt.  rend.  Acad.  d.  Sc.,  1909,  p.  157;  Ann.  de  1'inst.  Past.,  1910, 
1911,  1912. 

9  Anderson  and  Goldberger,  Jour.  A.  M.  A.,  1912,  p.  49;  Jour.  Med.  Res.,  1910, 
p.  469;   N.  Y.  Med.  Jour.,  1912,  p.  976. 

w  Rich-efts  and  Wilder,  Jour.  A.  M.  A.,  Feb.,  1910,  p.  463;  ibid.,  April  16, 
1910,  p.  1304;  ibid.,  April  23,  1910,  p.  1373;  ibid.,  July  23,  1910,  p.  309; 
Wilder,  Jour,  of  Inf.  .Dis.,  vol.  9,  1911. 

11  Gavino  and  Girard,  cited  from  Anderson  and  Goldberger. 


940  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

• 

is  followed  by  a  rapid  rise  of  temperature  after  an  incubation  time 
of  five  days  or  more,  and  the  fever  remains  high  for  three  to  five 
days,  after  which  it  comes  down  by  lysis.  Occasional  recrudescences 
have  been  noticed  in  monkeys.  Goldberger  and  Anderson  have  had 
a  mortality  of  2  per  cent  in  their  monkeys.  The  disease  may  be 
transmitted  from  monkey  to  monkey  with  the  blood,  which  is  in- 
fectious during  the  febrile  period  and  may  be  so  for  as  long  as 
thirty-two  hours  after  the  temperature  returns  to  normal. 

The  first  successful  transmission  of  the  disease  to  guinea-pigs 
was  accomplished  by  Ricketts  and  Wilder.  In  these  animals  the 
only  symptoms  are  fever,  a  matter  which  has  made  experimentation 
with  these  animals  relatively  difficult.  According  to  Nicolle,  guinea- 
pig  inoculation  may  occasionally  result  in  no  symptoms  at  all,  and 
yet  blood  of  such  animals  may  produce  the  fever  reaction  in  others 
inoculated  with  it.  There  seems  to  be  a  considerable  difference  in 
the  degree  of  susceptibility  in  guinea-pigs.  Anderson  found  about 
44  per  cent  of  his  guinea-pigs  resistant  in  the  first  generation  of 
transmission  from  the  typhus  patient.  Da  Rocha-Lima  believes  that 
about  80  to  90  per  cent  of  young  guinea-pigs  weighing  not  anore 
than  300  grams,  will  usually  be  found  susceptible.  The  typical  reac- 
tion to  guinea-pigs  is  a  rise  of  two  or  three  degrees  of  temperature 
on  the  sixth,  seventh  or  eighth  days. 

Although  ordinary  observation  shows  only  the  fever  reaction 
in  guinea-pigs,  it  has  been  lately  claimed  by  Lowy12  that  careful 
inspection  of  the  inner  surface  of  the  skin  of  guinea-pigs  may  reveal 
small  hemorrhagic  spots,  not  unlike  the  typhus  eruptions  in  human 
beings. 

Etiology. — The  disease  was  at  first  suspected  to  be  caused  by 
a  filtrable  virus,  an  opinion  which  is  still  held  by  some  observers. 
Most  workers  agree  to-day,  especially  because  of  the  work  of  Ander- 
son and  Goldberger,  that  filtered  blood  will  not  convey  the  disease, 
and,  although  Nicolle,  Conor  and  Conseil,  Ricketts  and  Wilder,  and 
others  have  reported  that  occasionally  inoculation  with  filtered  blood 
renders  monkeys  refractory  to  later  inoculation,  it  is  generally 
believed  at  present  that  the  disease  is  caused  by  some  agent  too 
large  to  pass  through  the  Berkefeld  or  Chamberland  filters. 

Work  on  the  etiology  of  typhus  has  been  very  extensive  and 
many  microorganisms  have  been  described. 


Lowy,  Wien.  klin.  Woch.,  18,  1916. 


TYPHUS  FEVER,   TRENCH  FEVER,  ETC.  941 

Ricketts  and  Wilder  saw  short  bacilli  in  smear  preparations,  but  were  not 
able  to  cultivate  them.  Rabinovitch13  described  a  Gram-positive  diplo-bacillus, 
cultivated  from  cases  of  an  epidemic  in  Kieff,  and  with  antigens  prepared 
from  this  organism,  he  obtained  complement-fixation  and  agglutination. 
Fiirth  studied  an  epidemic  in  China  and  obtained  short,  plump  rods  which 
grew  aerobically  in  short  chains.  P.  Th.  Muller  saw  a  diplo-bacillus  upon 
which  he  did  not  lay  much  stress  etiologically,  and  Prowezek  described  inclu- 
sions in  leucocytes  which  he  regarded  as  protozoa.  It  is  hardly  worth  while 
at  the  present  time  to  describe  in  detail  the  many  different  findings  that 
have  been  reported,  since  in  few  of  them  is  there  sufficient  evidence  to  enable 
us  to  come  to  conclusions. 

In  1914  Plotz14  described  a  short  Gram-positive  bacillus  which  he  obtained 
by  anaerobic  cultivation,  with  considerable  regularity,  from  cases  of  Brill's 
disease  at  the  Mt.  Sinai  Hospital,  New  York,  and  which  since  then  has  been 
made  the  subject  of  considerable  study  by  Plotz,  Olitsky,  and  Baehr.15  They 
have  obtained  the  bacillus  again  and  again,  have  succeeded  in  obtaining 
positive  agglutinations  and  complement-fixation  in  the  blood  of  endemic 
typhus  cases  after  the  crisis  and  have  obtained  a  similar  bacillus  from  a 
number  of  European  typhus  cases  which  have  come  into  quarantine. 

The  method  of  cultivation  by  which  this  bacillus  is  grown  is  relatively 
simple,  consisting  of  taking  blood  directly  from  a  vein  into  high  tubes 
containing  glucose  agar  and  unheated  and  unfiltered  ascitic  fluid  of  a  specific 
gravity  not  less  than  10.15. 

The  American  Red  Cross  Commission  which  went  to  Siberia  during  the 
last  typhus  epidemic — and  of  which  the  writer  was  a  member — attempted 
to  work  along  the  lines  laid  down  by  Plotz  but  found  it  extremely  difficult 
to  do  systematic  work  and  obtain  reliable  materials  under  the  conditions 
then  existing.  The  undersigned  obtained  an  organism  very  similar  to  the 
Plotz  bacillus  by  Plotz's  method  in  two  cases.  In  the  first  of  these  the 
organism  could  not  be  carried  further  than  the  second  generation,  and  in 
the  second  it  did  not  reach  America  alive.  Hopkins  obtained  a  similar 
organism  later  toward  the  end  of  the  epidemic.  However,  these  organisms 
were  found  so  rarely  that  we  were  forced  to  the  conclusion  that  these  isolated 
findings,  though  pointing  somewhat  in  favor  of  Plotz's  organism,  did  not 
establish  proof. 

Petruschky16  has  recently  cultivated  a  similar  but  aerobic  bacillus  from 
sputum  in  typhus  cases  and  Arnheim17  has  aerobically  cultivated  an  organism 
which  in  appearance  and  staining  properties  is  not  unlike  the  Plotz  bacillus. 

13  EaUnovitch,  Centralbk.  Bakt.  Orig.,  1909,  lii,  Arch,  f.  Hyg.,  1909. 

14  Plotz,  Jour,  of  A.  M.  A.,  Ixii,  20,  p.  14. 

15  Plots,  Olitsky  and  Baehr,  Jour,  of  Inf.  Dis.,  xvii,  1915,  p.  1. 

16  Petruschky,  Centralbk.  f.  Bakt.,  Ixxv,  1915,  p.  497. 
"Arnheim,  D.  Med.  Woch.,  36,  1916,  p.  1060. 


942  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

Arnheim  obtained  his  organism  from  six  eases,  on  ascitic  agar  plates,  on 
which  on  the  first  cultures  there  appeared  a  growth  hardly  visible  to  the 
naked  eye  and  which  in  transplants  continued  to  grow  very  delicately.  He 
states  his  organism  is  not  unlike  that  of  Petruschky  and  he  obtained  it  out 
of  the  blood,  the  sputum  and  the  urine  of  typhus  cases. 

A  serious  objection  to  the  acceptance  of  the  Plotz  bacillus  is  that, 
despite  the  fact  that  a  great  many  workers  have  been  studying  this 
disease  during  the  last  few  years,  the  microorganisms  which  have 
been  described  have  not  been  similar  one  with  the  other,  and  the 
fact  that  Plotz  organism  seems  to  lose  its  virulence  immediately  upon 
artificial  cultivation.  Also  according  to  Anderson,  active  immuniza- 
tion with  the  Plotz  bacillus  does  not  render  guinea-pigs  refractory  to 
virus  inoculation. 

Careful  experiments  at  the  Washington  Hygienic  Laboratory  also  have 
shown  that  the  inoculation  of  large  amounts  of  living  Plotz  organisms  will 
neither  injure  nor  immunize  guinea-pigs  or  monkeys,  whereas  a  single  injec- 
tion of  typhus  blood,  after  causing  the  typical  curve,  leaves  these  animals 
refractory  to  further  inoculation. 

In  the  course  of  the  war  a  great  deal  of  further  etiological  research  was 
done  on  typhus  incidental  to  which,  naturally,  renewed  search  for  bacterial 
causative  agents  was  made.  Gotschlich18  in  reviewing  this  work  finds  that 
only  two  other  investigators,  Paneth  and  Popoff,  found  organisms  similar 
to  the  Plotz  bacillus.  Gotschlich  believes,  as  we  do,  that  the  etiological 
relationship  of  the  Plotz  bacillus  has  by  no  means  been  proven  and  is  unlikely. 
The  indirect  evidence  adduced  by  Plotz  and  his  associates  with  agglutination 
reactions,  etc.,  has  lost  a  great  deal  of  its  value  in  view  of  the  more  recent 
work  done  on  the  Weil-Felix  reaction  in  which  a  species  of  proteus  is 
agglutinated  with  considerable  regularity  by  typhus  blood. 

It  is  quite  impossible  to  review  completely  the  enormous  bac- 
teriological literature  that  has  grown  up  about  claims  of  causative 
relationship  for  many  isolated  organisms.  In  none  of  them  could 
absolute  proof  be  adduced  and  such  claims  seem  to  become  less 
and  less  important  as  we  follow  the  more  recent  developments 
concerning  the  so-called  Rickettsia  bodies. 

In  1909  Ricketts19  saw  small  bacillus-like  bodies  in  the  blood 
of  guinea-pigs  he  had  infected  with  Rocky  Mountain  Spotted  Fever, 


18  Gotschlich,  Erg.  d.  Hyg.  Bakt.,  Berlin,  245,  1917. 
"Ricketts,  Medical  Record,  76,  1909,  842. 


TYPHUS  FEVER,   TRENCH  FEVER,   ETC.  943 

and  when  in  1910  he  studied  typhus  in  Mexico,  he  found  similar 
short  bacillus-like  forms  in  the  blood  of  typhus  fever  cases.  They 
were  extremely  small  and  stained  well  only  with  Giemsa.  In  1910, 
also,  Gavin  and  Girard20  saw  similar  bodies  in  the  blood  of  typhus 
cases  in  Mexico.  Prowazek21  working  in  Serbia  in  1910,  also  studied 
the  blood  of  typhus  fever  cases  and  saw,  within  leucocytes,  many 
small  rod  shaped  bodies,  not  unlike  those  described  by  Ricketts  and 
Wilder  and  by  Gavin  and  Girard.  In  1914  Sergent,  Foley  and 
Vialatte22  observed  similar  bodies  in  lice  taken  from  typhus  infected 
people,  and  this  was  confirmed  by  Nicolle,  Blanc  and  Conseil.28 

A  considerable  number  of  similar  observations  were  made 
by  other  workers  and  a  very  thorough  study  was  published  in  1916 
by  da  Rocha-Lima.24  Da  Rocha-Lima  found  these  small  bodies  in 
the  contents  of  the  alimentary  canals  of  lice  which  had  fed  on 
typhus  fever  patients.  At  first  he  did  not  find  similar  bodies  in 
lice  fed  on  normal  people,  and  he  definitely  concluded  that  these 
"organisms"  were  etiologically  related  to  the  disease  and  thought 
that  they  were  probably  protozoa.  It  was  he  who  suggested  that 
they  be  known  as  " Rickettsia-prowazeki"  in  honor  of  the  two  men 
who  had  died  in  the  study  of  the  disease.  Since  that  time  numerous 
investigations  have  been  published  by  da  Rocha-Lima,  Toepfer25 
and  others.  Among  the  most  important  investigations  that  have 
followed  are  those  of  Brumpt.26  Brumpt  obtained  evidence  that 
Rickettsia-like  bodies  could  be  found  in  lice  taken  from  healthy 
individuals  and  that  these  organisms  could  remain  in  lice  throughout 
the  entire  life  of  the  louse,  while  the  typhus  virus  did  not  seem 
to  be  active  in  the  lice  for  longer  than  about  eight  or  nine  days. 
Arkwright,  Bacot  and  Duncan27  using  lice  bred  from  a  clean  stock 
and  working  with  trench  fever,  showed  that  Rickettsia-like  bodies 
could  be  found  in  the  lice  after  feeding  on  trench  fever  patients. 
Strong  has  tabulated  on  pages  77  to  80  of  the  Red  Cross  Report 
referred  to  above,  all  the  various  observations  that  have  been  made 
upon  Rickettsia-like  bodies  in  lice  in  connection  with  various  dis- 

30  Gavin  and  Girard,  Bull,  do  1'Inst.  Past.,  8,  1910,  841. 

21  Prowazek. 

22  Sergent,  Foley  and   1'ialalte,  Compt,  rend,  de  la  Soc.  Biol.,  77,  1914,  101. 
"Nicolle,  Blanc  and  Conceit,  Compt.  rend,  de  la  Acad.  de  Sciences,  159,  1914. 
24  da  Rocha-Lima,  Berl.  klin.  Woch.,  2.1,  1916,  and  Munch,  ined.  Woch.,  39,  1916. 
26  Toepfer,  Berl.  klin.  Woch.,  515,  1910,  323,  and  Med.  Klinik,  1.3,  1917,  678. 

26  Brumpt,  quoted  from  Strong,  loc.  cit. 

"Arkwright,  Bacot  and  Duncan,  Proe.  Roy.  Soc.  Med.,  13,  1919,  23. 


944  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

eases.  It  appears  from  this  literature  that  Rickettsia-like  micro- 
organisms may  be  present  in  lice  fed  upon  healthy  people,  as  well 
as  in  lice  fed  upon  typhus  cases,  upon  trench  fever  and  upon  healthy 
individuals.  Recent  and  important  investigations  by  Wolbach,  Todd 
and  collaborators  seem  to  show  that  all  these  observations  are 
correct,  but  that  it  is  likely  that  the  R.  prowazeki  associated  by 
da  Rocha-Lima  with  typhus  differs  materially  from  some  of  the 
other  Rickettsia  bodies. 

All  this  elaborate  work  leaves  us  still  considerably  in  the  dark. 
It  is  not  at  all  conclusively  definite  that  the  Riekettsia  bodies  are 
microorganisms,  but  those  who  have  studied  them  most  carefully 
seem  to  feel  reasonably  sure  that  they  are.  Since  the  organisms 
cannot  be  cultivated,  positive  differentiation  between  the  various 
observed  forms  is  not  possible.  The  finding,  however,  of  similar 
appearances  in  the  blood  of  typhus  patients  and  in  tissue  sections 
gives  a  certain  amount  of  basis  for  tentative  etiological  suggestions. 
In  a  separate  section  taken  largely  from  Wolbach 's  description,  we 
have  given  the  general  characterization  of  Rickettsia  bodies  as  a 
class.  The  R.  prowazeki  of  da  Rocha-Lima,  like  the  others,  is  very 
small,  being  rarely  more  than  two  micra  in  length,  and  often  much 
less,  is  non-motile,  is  hard  to  stain,  gives  a  reddish-blue  color  with 
Giemsa  and  is  found  intracellularly  particularly  in  the  gastric  and 
intestinal  epithelium  of  the  mouse.  In  typhus  patients  we  have 
already  mentioned  the  similar  bodies  seen  by  Ricketts  and  Wilder 
in  the  blood,  and  Prowazek  found  similar  bodies  within  the  leu- 
cocytes of  typhus  blood.  This  has  been  confirmed  by  Lipschiitz 
and  others. 

Attempts  to  identify  the  Plotz  bacillus  and  other  organisms  with 
the  Rickettsia  do  not  seem  logical  at  the  present  time  since  the 
Plotz  bacillus  can  be  cultivated  with  relative  ease,  while  Rickettsia 
bodies  cannot  be  cultivated.  It  is  very  difficult  to  stain  them,  and 
they  decolorize  by  Gram,  The  painstaking  work  of  Wolbach,  Todd 
and  their  associates  is  not  yet  published.  It  will  be  very  shortly 
published,  however,  in  Wolbach 's  Harvey  Lecture  and  the  reader 
is  referred  to  this  article. 

Prevention  of  Typhus  Fever. — From  what  has  been  said  above, 
it  is  plain  that  the  prevention  of  typhus  fever  must  be  centered 
chiefly  upon  delousino-,  both  of  patients  and  of  the  population  as 
a  whole.  Delousirig  methods  will  be  dealt  with  in  speaking  of  the 
general  prevention  of  louse-borne  diseases  at  the  end  of  this  section. 


TYPHUS  FEVER,   TRENCH   FEVER,   ETC.  945 

Immunity. — A  single  attack  of  typKus  fever  seems  to  protect 
permanently.  Monkeys  and  guinea-pigs  that  have  once  passed 
through  the  febrile  reaction  are  thereafter  refractory. 

Prophylactic  vaccines  made  from  various  bacteria  isolated  from 
typhus  cases  have  not  in  our  opinion  given  convincing  proof  of 
success.  Attempts  to  immunize  prophylactically  with  inactivated 
typhus  blood  have  not  so  far  had  sufficient  test.  Specific  therapy 
with  the  serum  of  convalescents  attempted  by  Nicolle  and  others 
have  not  yet  borne  sufficient  fruit  to  warrant  very  much  hope. 

The  Weil-Felix  Reaction. — This  reaction  is  of  peculiar  interest 
in  that  it  represents  a  diagnostic  serum  reaction  in  typhus  with  an 
organism  which  quite  surely  has  no  etiological  relationship  to  the 
disease.  It  was  first  described  by  Weil  and  Felix28  in  1916,  after 
isolation,  from  the  urine  of  a  case  of  typhus  fever>  of  an  organism 
which  agglutinated  in  the  serum  of  the  patient  in  a  dilution  of 
1 :200.  The  organism  apparently  belonged  to  the  proteus  type  and 
was  designated  by  them  as  "  Proteus  X2. "  Further  study  with  it 
showed  that  similar  cases  also  agglutinated  this  organism.  Later 
another  bacillus  X19,  very  similar  to  the  first  one,  was  isolated  from 
another  case. 

The  organism  is  a  Gram-negative,  motile  bacillus  which  ferments 
glucose  and  lactose,  coagulates  milk  with  acid  formation,  liquefies 
gelatin  and  in  colony  appearance  resembles  the  proteus  group. 
Bengston29  of  the  United  States  Public  Health  Service  has  studied 
the  two  organisms  ("X2"  and  "X19"  of  Weil  and  Felix)  bac- 
teriologically,  and  has  compared  them  to  laboratory  cultures  of 
proteus.  She  found  that  they  were  very  slow  gelatin  liquefiers, 
that  they  were  extremely  slow  in  digesting  coagulated  blood  serum, 
but  that  they  did  not  ferment  lactose.  In  this,  the  organisms  of 
Weil  and  Felix  which  she  studied  resembled  one  reported  by  Fair- 
ley.30  Agglutination  reactions,  against  proteus  sera  produced  with 
other  strains,  were  in  some  cases  active  against  these  strains,  and 
conversely  sera  produced  with  the  Weil-Felix  strains  were  more  active 
against  some  other  proteus  organisms. 

Apparently  the  agglutination  of  proteus  "X"  strains  is  of  dis- 
tinct value  in  typhus  diagnosis.  Fairley  observed  positive  agglutina- 
tion of  these  organisms  in  97  per  cent  of  the  typhus  sera  which 

28  Weil  and  Felix,  Wien.  klin.  Woch.,  No.  2,  1916. 

29  Bengston,  Jour.  Infec.  Dis.,  24,  1919,  428. 

30  Fairley,  Jour,  of  Hyg.,  18,  ]9]9,  203. 


946  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

he  examined.  He  never  obtained  agglutination  with  the  blood  of 
non-typhus  cases,  using  dilutions  of  1 :20  in  all  the  tests.  Weil 
and  Felix  found  that  almost  all  their  clinically  typical  typhus  cases 
agglutinated  this  organism,  whereas  very  few  sera  of  non-typhus 
patients  and  no  normal  sera  showed  agglutination  in  dilutions  of 
1 :25.  Fairley  repored  that  of  thirty-five  cases  examined  during  the 
febrile  stage,  all  agglutinated  the  organisms  in  dilutions  of  1 :20 
and  1 :1200  after  the  fifth  day  and  throughout  the  second  week. 

The  test  is  carried  out  by  growing  the  organisms  on  agar,  sus- 
pending them  in  salt  solution  and  testing  with  dilutions  of  1 :25  and 
1  :50  of  the  serum  of  suspected  cases.  The  agglutination  titer  in 
true  typhus  cases  may  rise  as  high  as  1 :200  or  higher  by  the  end 
of  the  second  week.  Bengston  states  that  in  one  test  made  on  a 
typhus  case  there  was  complete  agglutination  of  the  Weil-Felix 
organisms  in  diluton  of  1 :400,  while  cultures  of  the  Rawlings  typhoid 
and  of  Proteus  vulgaris  were  not  agglutinated  in  dilutions  of  1 :50. 

The  explanation  of  this  reaction  is  doubtful.  It  may  be  assumed 
quite  definitely  that  this  organism  has  no  etiological  relationship 
to  typhus.  It  is  possible  that  in  typhus  fever  secondary,  non-specific 
agglutinating  antibodies  for  a  variety  of  organisms  may  be  present. 
We  need  only  call  attention  to  the  antibody  reactions  carried  out 
with  the  Plotz  bacillus  and  with  some  of  the  other  organisms  for 
which  etiological  relationship  has  been  claimed  in  this  connection. 
Incidentally,  the  Weil-Felix  reaction  adds  to  our  scepticism  about 
Plotz 's  claims. 


TRENCH  FEVER  (WOLHYNIAN  FEVER) 

In  1919  there  appeared  among  the  Armies  at  the  front  a  disease 
which  did  not  clinically  resemble  the  ordinary  well-known  febrile 
diseases.  Cases  of  this  condition  were  seen  among  British  troops 
by  Graham  and  Herringham31  and  on  the  German  front  in  Poland 
and  Wolhynia  similar  ones  were  described  by  His32  and  by  WTerner.33 
Apparently  the  disease  had  been  noticed  by  Gratzer34  as  early  as 
1914.  Cases  appeared  in  enormous  numbers  and  because  the  disease 

31  Graham,  Lancet,  2,  1915,  703;  Herringham,  Lancet,  9,  1916,  429. 

32  His,  Berl.  klin.  Woch.,  53,   1916,  738. 

33  Werner,  Munch.  Med.  Woch.,  63,  402,  1916. 
»4  Gratzer,  Wien.  klin.  Woch.,  29,  295,  1916. 


TYPHUS  FEVER,   TRENCH  FEVER,  ETC.  947 

seemed  to  arise  almost  entirely  from  the  front  areas  was  spoken  of 
as  Trench  fever. 

The  Disease. — Work  on  the  clinical  differentiation  of  the  disease 
was  done  by  a  great  many  army  surgeons.  An  accurate  report  was 
made  by  McNec,  Brunt  and  Renshaw,;i5  by  a  number  of  German 
workers,  and  finally  by  a  British  and  by  an  American  commission, 
the  American  group  organized  under  Strong,  and  including  as 
clinician  Homer  Swift.  In  a  Harvey  Lecture  by  Swift36  printed  in 
the  Archives  of  Internal  Medicine,  July,  1920,  an  accurate  summary 
of  available  facts  concerning  this  disease  to  date  may  be  found. 

The  disease  is  sudden  in  onset  with  fever,  headache  and  pains 
in  the  muscles.  The  onset  resembles  that  of  influenza.  In  a  few 
days,  pain  and  tenderness  of  the  joints  appears,  and  the  temperature 
shows  peculiar  remissions  which  Swift  characterizes  as  being  of  the 
" spiky"  type. 

Characteristic  of  the  disease  are  the  bone  pains  which  are  not 
accompanied  by  any  signs  of  inflammation.  There  may  be  continued 
hyperesthesia.  There  may  be  sensory  disturbances  with  increase  of 
the  tendon  reflexes.  The  fever  curves  are  very  irregular,  some  show- 
ing the  intermittent  "spiky"  type  referred  to  above,  others  develop- 
ing the  typhoid-like  ladder  type.  Another  characteristic  is  the 
frequency  of  relapses,  in  which,  after  remissions  of  varying  intervals, 
a  second  rise  of  temperature  comes  on.  The  relapses  may  come  on 
after  weeks  or  months.  A  case  of  which  we  have  personal  knowledge 
has  developed  two  relapses  in  the  course  of  two  years  after  the 
original  attack.  In  other  cases  Swift  states  that  the  manifestations 
may  assume  a  subacute  or  chronic  form  with  low  grade  fever  which 
may  continue  for  months. 

Transmission  and  Etiology. — McNee,  Brunt  and  Renshaw,  in 
1916,  succeeded  in  transferring  the  disease  from  man  to  man  by 
intravenous  and  intramuscular  injections  of  whole  blood.  In  these 
early  experiments  they  found  that  the  plasma  if  entirely  free  from 
hemoglobin,  was  not  infectious,  but  that  the  red  cells  contained  the 
virus  even  after  repeated  washings.  They  did  not  succeed  in  passing 
the  virus  through  a  Berkefeld  filter.  In  1917,  Werner,  whom  we 
quote  from  Swift,  allowed' himself  to  be  bitten  by  lice  that  had 
previously  fed  on  trench  fever  patients,  and  is  said  to  have  con- 


KMcNee,  Brunt  and  Kenshaw,  Brit.  Med.  Jour.,  1,  1916,  295. 
"Swift,  Harvey  Lecture,  Harvey  Soc.,  New  York,  Jan.  10,  1920. 


948  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

traded  a  mild  form  of  the  disease.  A  similar  observation  was  made 
by  Kuczynski37  oil  himself.  Davies  and  Weldon38  in  the  same  year 
carried  out  a  similar  experiment,  allowing  themselves  to  be  bitten 
by  lice  immediately  after  the  lice  had  fed  on  trench  fever  patients. 
One  of  them  developed  trench  fever  twelve  days  later.  A  similar 
experiment  on  a  volunteer  was  successfully  made  by  Pappenheimer 
and  Mueller39  in  1917,  but  in  criticising  all  these  experiments  Swift 
believes  that  the  proof  brought  was  not  sufficiently  conclusive  be- 
cause of  inadequate  control.  In  1918  two  commissions  were  formed 
for  the  purpose  of  studying  the  disease.  The  American  commission 
was  aided  by  a  British  entomologist,  Captain  Peacock.  The  first 
result  of  these  investigations  was  that  McNee's  observation  about 
transmission  with  whole  blood  was  confirmed,  but  it  was  found, 
in  contrast  to  McNee's  results,  that  the  plasma  as  well  as  the  red 
blood  cells  was  infectious.  It  seemed  that  the  blood  taken  as  early 
as  the  fourth  day  of  the  disease  was  more  infectious  than  that 
obtained  later.  Transmission  after  filtration  through  Berkefeld 
filters  did  not  succeed  at  first,  but  Swift  records  that  later,  when 
infectious  urine  was  used,  filtration  was  successful.  It  was  found 
at  this  time  that,  as  well  as  the  blood,  the  urine  jf  patients  is  also 
infectious. 

Careful  experiments  were  made  with  lice,  all  of  which  were 
reared  from  eggs  and  fed  on  normal  subjects,  and  the  non-infectious- 
ness  of  these  lice  was  proven  by  allowing  them  to  feed  on  eleven 
different  uninfected  people.  Such  lice  were  allowed  to  feed  several 
times  on  trench  fever  patients  and  subsequently  allowed  to  feed 
on  twenty-three  volunteers,  78  per  cent  of  whom  developed  trench 
fever.  It  did  not  seem  necessary  for  the  lice  to  be  in  contact  with 
the  skin  while  feeding,  nor  was  it  necessary  to  produce  scarification. 
In  two  instances  the  mere  bite  of  the  louse  through  the  meshes 
of  gauze  covering  the  box  produced  the  disease.  The  incubation 
time  in  these  louse  transmitted  cases,  varied  from  fourteen  to  thirty- 
eight  days,  the  average  being  twenty-one.  Meanwhile,  the  British 
commission  found  that  the  excreta  of  infected  lice  applied  to  scari- 
fied skin  could  also  produce  the  disease,  thus  showing  that  the 


87  KuczynsTci,  Eeported  from  Jungmann,  Deut.  med.  Woeh.,  64,  1917,  359. 
38  Davies  and  Weldon,  Lancet,  1,  1917,  183. 

"Pappenheimer  and  Mueller,   Amer.   Red   Cross  Committee  Report,  London, 
1918,  Oxford  Press. 


TYPHUS   FEVER,   TRENCH   FEVER,   ETC.  949 

excrement  of  lice  that  have  bitten  trench  fever  patients  may  be 
infectious  when  it  comes  in  contact  with  lesions  on  the  skin. 

Byam40  showed  that  as  late  as  300  to  400  days  after  the  onset 
of  the  disease,  trench  fever  patients  can  still  infect  lice.  This  is  of 
great  importance  in  appraising  the  epidemiological  possibilities  of 
carriers.  Lice  could  also  be  infected  by  patients  during  the  periods 
of  remission. 

Both  the  British  and  the  American  commission  showed  that  the 
virus  is  probably  not  transmitted  through  the  eggs  of  the  louse. 
The  British  commission  reported  that  the  headlouse  can  transmit 
the  disease  through  its  excreta  in  the  same  way  as  the  body  louse. 
Other  insects,  however,  did  not  seem  to  carry  the  disease. 

As  to  the  causative  agent,  little  is  definitely  known.  Toepfer,41 
da  Rocha-Lima42  and  other  German  observers  who  have  studied 
Rickettsia  bodies  in  typhus  fever  were  encouraged  to  undertake 
similar  studies  in  connection  with  trench  fever  because  of  the 
similarity  of  the  means  of  conveyance  of  the  two  diseases.  These 
observers,  as  well  as  Jungmann43  succeeded  in  finding  Rickettsia 
bodies  in  the  intestines  of  lice  fed  on  trench  fever  patients.  Da 
Rocha4jima  comparing  lice  that  had  bitten  individuals  who  did  not 
have  trench  fever  with  those  fed  on  trench  fever  patients  found 
that  72  per  cent  of  the  insects  found  on  the  trench  fever  patients 
showed  Rickettsia  bodies,  but  20  per  cent  of  those  fed  on  normal 
people  showed  similar  ones.  Arkwright,  Bacot  and  Duncan44  found« 
similar  Rickettsia  bodies  in  a  large  number  of  lice  that  had  fed 
several  times  on  sixty-four  trench  fever  patients.  They  found  the 
bodies  in  only  one  out  of  many  lots  of  insects  fed  on  normal  people. 
Their  experiments  seem  to  indicate  that  when  Rickettsia  bodies 
appear  in  the  excrement  of  lice  after  feeding,  these  excrements  were 
infectious. 

It  is  quite  clear  that  no  positive  conclusions  can  be  drawn, 
especially  in  view  of  the  frequent  finding  of  Rickcttsia-like  bodies 
in  lice  that  have  had  no  connection  with  this  disease.  The  clue 
furnished  by  the  finding  of  Rickettsia,  however,  must  be  followed, 

40  Byam,  et  al.,  Trench  Fev?r,  Brit.  War  Office  Commit.  Eep.,  Oxford  Press, 
1919;  Bycm,  Proc.  Roy.  Soc.  Med.,  13,  1919,  19. 

41  Toepfer,  Munch,  mod.  Woch.,  03,   1916,  1495. 

42  da  It ocha- Lima,  Munch,  mod.  Woc-h.,  04,  1917. 
"Jungmann,  Dent,  med.  Woch.,  64,  1917,  359. 

"Arkwright,  Bacot  and  Duncan,  Proc.  Koy.  Soc.  Med.,  13,  1919,  23. 


950  DISEASES  CAUSED   BY   F1LTRABLE   VIRUS 

since  the  possibilities  here  are  the  only  ones  that  seem  to  show 
great  etiological  promise  at  the  present  time. 

Prevention  of  trench  fever,  like  the  prevention  of  typhus,  depends 
upon  delousing. 

ROCKY   MOUNTAIN   SPOTTED   FEVER 

Rocky  Mountain  Spotted  Fever  is  a  disease  which  has  long 
existed  in  the  United  States.  A  thorough  review  of  the  entire 
subject  has  been  made  by  Wolbach45  who  states  that  authentic  cases 
were  reported  as  early  as  1873.  The  disease  has  been  pretty  well 
limited  to  the  mountainous  regions,  most  of  the  cases  being  reported 
from  Idaho  and  Montana. 

The  Disease. — The  onset  of  the  disease  is  usually  abrupt,  though 
occasionally  it  may  be  preceded  by  a  few  days  of  general  malaise. 
It  not  infrequently  begins  with  a  chill,  followed  by  a  rapid  rise  of 
temperature  which  reaches  102°  or  104°.  The  temperature  may 
show  morning  remissions  with  rises  of  one  or  two  degrees  in  the 
evening,  and  gradually  increasing,  reaching  its  height  during  the 
second  week.  On  the  third  or  fourth  day  after  the  onset  of  the 
disease,  a  rash  appears  first  on  the  wrists,  ankles  and  back,  later 
upon  the  arms,  legs  and  chest,  extending  to  the  forehead  and 
abdomen.  It  is  always  least  marked  on  the  abdomen,  according 
to  Wolbach.  It  comes  out  in  the  course  of  about  thirty-six  hours 
and  may  also  involve  the  mucous  membranes  of  the  mouth  and 
pharynx.  The  temperature  remains  up  after  the  appearance  of  the 
rash!  The  rash  consists  of  little  red  patches  about  4  or  5  mm.  in 
diameter  which  at  first  disappear  on  pressure.  Like  the  typhus 
rash,  they  become  darker  red,  then  purplish  and  later  hemorrhagic 
in  character.  Small  petechial  spots  may  appear  in  the  center.  They 
fade  into  pigmented  spots  later.  During  recovery  desquamation 
occurs. 

There  may  be  violent  nervous  symptoms.  The  blood  picture  is 
not  altered  materially,  the  leucocytes  slightly  increase  in  number, 
but  the  differential  count  remains  approximately  normal. 

The  mortality  of  the  disease  as  estimated  from  various  sources 
by  Wol bach  for  1915  and  1916,  ranged  between  7  and  13  per  cent. 

The  incubation  time  seems  to  vary  between  three  and  nine  days. 


45  Wolbach,  Jour.  Med,  Kes.,  41,  1919,  1. 


TYPHUS   FEVER,   TRENCH  FEVER,   ETC.  951 

Epidemiology. — The  distribution  of  the  disease  follows  that  of 
the  wood  tick,  Dermacentor  venustus.  The  disease  occury  in  Idaho, 
Montana,  Nevada,  Wyoming,  California,  Colorado  and  Washington. 
Wolbach  notes  that  the  distribution  of  cases  in  various  states  seems 
to  be  restricted  to  definite  localities.  In  Idaho,  he  finds  that  the 
cases  are  particularly  frequent  in  the  Snake  River  Valley  and  in 
Montana  in  the  Bitter  Root  Valley  where  there  seem  to  be  infectious 
foci.  Seasonally  the  disease  occurs  almost  entirely  in  the  spring. 

The  wood  tick  named  above  was  associated  with  the  disease  first 
by  Wilson  and  Chowning40  in  1902.  RickeUs  brought  proof  of  this 
in  1906,47  showing  that  the  disease  could  be  produced  in  guinea-pigs 
by  allowing  wild  ticks  of  this  species  to  feed  upon  them.  McClintic48 
confirmed  this  and  his  investigations  with  those  of  Ricketts,  cen- 
tralized attention  upon  this  particular  species,  the  Dermacentor 
venustus.  Larvae,  fed  upon  infected  ticks,  remain  infective  into 
the  nymph  stage  and  the  nymph  once  infected  remains  infected  into 
the  adult  stage.  He  also  showed  that  eggs  from  infected  females 
would -produce  the  disease  when  injected  into  guinea-pigs  and  that 
both  male  and  female  ticks  would  transmit  it.  Wolbach 's49  inves- 
tigations have  confirmed  most  of  these  points. 

Guinea-pigs  infected  by  ticks  develop  a  temperature  in  about 
three  to  seven  days.  Injected  with  blood  from  other  guinea-pigs, 
the  disease  may  begin  at  the  end  of  forty-eight  hours.  Death,  which 
often  follows,  occurs  on  the  sixth  or  seventh  day.  As  the  disease 
progresses  there  is  swelling  and  reddening  of  the  skin  of  the  scrotum, 
loss  of  appetite  and  general  signs  of  illness.  There  may  be  redness 
and  swelling  of  the  eye-lids,  ears  and  paws,  and  ulcers  of  the  paws 
may  form.  On  autopsy,  there  may  be  edema  and  hemorrhages  of 
the  skin  and  subcutaneous  tissues  of  the  scrotum.  Male  guinea-pigs 
show  the  disease  most  characteristically  because  of  the  scrotal  lesions. 
Rabbits  are  susceptible,  although  not  regularly  so.  Foot50  has 
studied  this  under  Wolbach 's  direction.  When  it  does  occur 
in  rabbits,  the  disease  is  virtually  the  same  as  that  occurring  in 
guinea-pigs,  except  that  in  addition  to  the  other  signs  of  illness, 


49  Wilson  and  Chowning,  Jour.  A.  M.  A.,  39,  1902. 

47  Bicketts,  Jour.  A.  M.  A.,  46,  1906. 

48  McClintic,    U.    S.    Pub.   Health    and    Marino   Hosp.    Serv.,   Weekly    Bulletin, 
.  20,  27,   1912. 

•"•  Wolbach,  Jour.  Med.  Research,  41,  1919-1920,  3. 
,  Jour    Med.,  Res.,  39,  1919. 


952  DISEASES  CAUSED  BY  FTLTRABLE  VIRUS 

the  ears  are  often  inflamed,  and  in  white  rabbits  thrombosed  vessels  in 
the  ears  can  be  seen. 

Monkeys  are  susceptible  arid  usually  die  at  the  end  of  seven  days. 

Etiology. — The  lesions  in  the  blood  vessels  in  animals  and  man 
indicate  the  presence  of  the  causative  agent  in  these  locations.  Wol- 
bach49  who  studied  histological  material  from  this  point  of  view, 
found  within  the  endothelial  cells  of  the  vascular  lesions,  in  smooth 
muscle  cells  of  the  media,  as  well  as  occasionally  in  detached  en- 
dothelial cells  present  in  thrombi  extremely  minute  small  "diplo- 
coccmlike"  organisms.  These,  he  states,  stain  with  eosin-methylene- 
blue.  The  organisms  were  best  stained  with  Giemsa  solution  with 
which  they  appear  as  slender  pale  blue  rods.  There  was  a  distinct 
contrast  between  the  appearance  of  these  organisms  and  that  of 
accidentally  introduced  bacteria,  in  that  the  shape  of  the  former 
was  vaguely  outlined  and  less  sharp  than  that  of  the  bacteria. 
Ricketts51  had  described  similar  organisms  in  the  blood  of  man  and 
guinea-pigs  which  he  described  as  "lanceolate  chromatin-staining 
bodies"  in  sets  of  two,  a  small  amount  of  eosin-staining  substance 
appearing  between  the  two  individuals.  The  same  organism  was 
seen  in  smears  of  the  intestinal  contents  of  ticks.  Wolbach  has  also 
seem  them  in  smear  preparations  made  from  the  eggs  of  infected 
ticks. 

In  the  case  of  these  bodies,  as  well  as  in  those  described  in 
connection  with  typhus  fever,  definite  conclusions  cannot  be  reached 
as  yet.  A  careful  analysis  of  the  entire  subject  will  be  found  in 
Wolbach 's  paper  of  1919.  The  organisms,  if  they  are  organisms, 
probably  belong  to  the  class  of  Rickettsia.  They  have  not  been 
cultivated. 

The  prevention  of  the  disease  depends  largely  upon  the  preven- 
tion of  tick  bites  and  the  suppression  of  the  wood  tick,  a  matter 
which  is  very  difficult  in  the  countries  in  which  they  abound. 

51  Eicketts,  Jour.  A.  M.  A.,  47,  1906,  33 ;  Jour.  A.  M.  A.,  47,  1906,  358  ;  Jour. 
A.  M.  A.,  47,  1906,  1067;  Jour.  A.  M.  A.,  49,  1907,  1278;  Jour.  Infec.  Dis.,  4, 
1907,  141;  Jour.  A.  M.  A.,  49,  1907,  24;  Trans.  Chicago  Path.  Soc.,  1907;  Jour. 
Infec.  Dis.,  5,  1908,  221;  Jour.  A.  M.  A.,  52,  1909,  379;  Medical  Record,  76, 
1909,  842. 


TYPHUS  FEVER,   TRENCH  FEVER,  ETC,  953 

LICE    AND    DELOUSING 

The  sanitation  of  typhus  fever,  of  trench  fever  and  of  some  forms 
of  relapsing  fever  is  so  definitely  dependent  upon  processes  of  louse 
extermination  that  a  few  paragraphs  on  the  habits  of  lice  and  the 
means  for  their  destruction  will  contain  the  most  important  prin- 
ciples upon  which  sanitary  efforts  in  the  prevention  of  these  diseases 
must  be  based.  The  lice  which  infest  the  human  body  are  of  two 
types,  the  Pediculus  humanus  which  includes  the  body  louse  and  the 
head  louse,  and  the  Phthyrius  pubis,  or  the  pubic  or  crab  louse.  It 
is  the  first  two  of  these,  the  body  louse  and  the  head  louse  with  which 
sanitarians  are  most  concerned. 

For  an  anatomical  description  of  lice  we  must  refer  the  reader 
to  Nuttall's  comprehensive  Monograph  in  the  British  Journal  of 
Hygiene  of  1917,  and  to  textbooks  on  medical  entomology.  The 
following  facts,  important  for  the  sanitarian,  are  compiled  from 
various  sources. 

The  ordinary  life  of  a  louse  is  about  four  to  six  weeks.  The 
female  louse  begins  to  lay  eggs  about  eight  or  nine  days  after 
hatching.  It  is  stated  that  such  lice,  well  fed,  and  in  normal  en- 
vironment, will  lay  altogether  about  300  eggs  at  the  rate  of  ten 
or  so  a  day.  It  takes  about  one  week  to  eight  days  for  these  eggs 
to  hatch.  On  this  basis,  a  single  generation  of  lice  takes  about 
sixteen  to  eighteen  days. 

The  louse  prefers  to  lay  its  eggs  upon  little  threads  or  hairs, 
more  readily  upon  rougher  cloth  than  upon  silk.  It  was  suspected 
in  the  early  part  of  the  war  that  silk  underclothing  gave  some 
protection,  but  apparently  this  is  not  of  very  much  use.  The  body 
louse  prefers  to  lay  its  eggs  on  the  inner  surfaces  of  underclothing 
and  other  clothing,  preferably  along  the  seams,  and  on  blankets, 
though  most  of  the  louse  eggs  are  probably  laid  on  underclothing. 
It  should  not  be  forgotten  that  in  arranging  for  disinfestation,  the 
outer  clothing,  overcoats,  blankets,  etc.,  may  also  be  infested.  In 
addition  to  this,  both  the  head  louse  and  the  body  louse  may  lay 
their  eggs  on  the  hairs  of  the  body.  The  louse,  being  an  habitual 
parasite  on  animals  and  man,  prefers  to  lay  its  eggs  at  a  temperature 
little  below  that  of  the  body,  a  temperature  which  is  stated  as 
ranging  about  30°  C.  If  the  temperature  is  lower  than  that,  it 
takes  them  two  weeks  or  more  to  hatch.  According  to  studies  made 
by  British  Army  sanitarians,  they  will  not  hatch  below  22°  C.  and 


954  DISEASES  CAUSED  BY  FILTKABLE  VIRUS 

the  most  favorable  temperature  for  hatching  is  at  35°  C.,  when  the 
hatching  time  is  very  much  speeded  up  and  may  be  less  than  eight 
days.  It  is  important  to  know  that  louse  eggs  are  destroyed  by 
temperatures  slightly  above  60°  C.  But  it  is  not  safe  to  rely  upon 
such  low  temperatures  for  disinfestation.  While  the  nits  are  quite 
susceptible  to  temperature,  they  are  much  harder  to  destroy  by 
insecticides  than  are  the  adult  lice.  It  is  important  to  remember 
that  many  of  the  insecticide  substances  which  are  applied  to  the 
body  and  clothing  for  the  prevention  of  lousiness  may  keep  lice 
away  but  will  not  kill  them  when  once  present. 

It  is  also  important  to  remember  that,  although  the  adult  louse 
must  feed  with  some  regularity  in  order  to  thrive  and  lay  its  eggs, 
the  eggs  may  remain  alive  on  clothing,  underwear,  etc.,  for  a  month 
at  least,  away  from  the  human  body,  and  may  be  hatched  out  when 
this  clothing  is  put  on.  Thus,  clothing,  underwear,  blankets,  etc., 
of  louse  infested  dugouts,  huts,  ships,  etc.,  must  be  taken  care  of 
even  if  it  has  not  been  worn  for  some  time. 

Although  the  louse,  like  the  bed  bug  in  the  song,  has  no  wings  * '  at 
all,"  and  is  not  a  wanderer,  it  is  astonishing  how  easily  it  can  pass 
from  one  individual  to  another.  Lice  may  be  easily  acquired  during  the 
examination  of  a  case,  in  passing  through  a  crowd,  or  in  handling 
underwear  and  clothing  in  laundry  work  or  disinfesting  operations. 

The  adult  louse  feeds  about  twice  a  day,  and  the  louse  bites, 
while  they  may  be  quite  annoying  to  some  individuals,  may  cause 
practically  no  reaction  or  annoyance  in  habitually  lousy  persons. 
They  are  apt  to  leave  the  body  of  the  sick  and  usually  do  leave 
the  body  of  the  dead  as  soon  as  possible.  When  removed  from 
human  sources  of  food,  they  may  die  in  anywhere  from  one  or  two 
days  to  a  week.  Nine  or  ten  days  is  stated  as  the  probable  limit 
to  which  the  adult  may  live  in  clothing  that  has  been  hung  up  or 
put  away. 

Delousing  depends  upon  early  discovery  of  lousiness  in  a  com- 
munity, regiment  or  other  unit,  personal  cleanliness,  disinfestation 
of  those  who  are  lousy  and  disinfestation  of  clothing,  blankets,  etc., 
and  quarters. 

In  armies  and  in  communities  during  the  existence  of  louse-borne 
diseases,  inspection  for  lousiness  of  bodies,  underclothing,  etc., 
should  be  carried  out  at  frequent  intervals.  The  individual  who 
attempts  to  protect  himself  should  inspect  his  own  body  and  under- 
clothing on  going  to  bed  at  night. 


TYPHUS  FEVER,   TRENCH   FEVER,   ETC.  955 

The  most  efficient  individual  protection  against  louse  infestation 
consists  in  frequent  baths,  preferably  hot  showers,  in  which  a  free 
use  of  soap  is  made,  and  all  the  hairy  parts  of  the  body  very 
thoroughly  soaped.  The  best  type  of  soap  is  a  soft  soap.  A  British 
Army  preparation  which  was  very  useful  during  the  war  was  made 
by  slowly  warming  three  pounds  of  soft  soap  with  one-half  pint 
of  water  and,  after  removal  from  the  fire,  this  was  mixed  with 
five  and  one-half  pounds  of  crude  paraffin  oil.  Two  and  one-half 
per  cent  cresol  was  added  to  this  mixture.  This  formula  is  taken 
from  D.  G.  M.  S.  Circular  Memorandum,  No.  16,  of  the  British  Army. 
After  bathing,  a  complete  change  of  underclothing  should  be  made. 
In  the  American  Army  bathing  establishments  were  arranged  from 
ordinary  Adrian  huts,  which  were  applicable  to  delousing  on  a  large 
scale.  Bath  houses  were  so  arranged  that  men  undressed  in  an 
anteroom,  tying  up  their  outer  clothing  into  bundles  with  tags 
attached  and  throwing  their  soiled  underclothing  into  large  wire 
baskets  which  were  immediately  taken  to  the  steam  sterilizers.  They 
then  passed  into  the  shower  rooms  and  came  out  into  a  dressing 
room,  into  a  window  of  which  the  outer  clothing  after  sterilization 
was  returned  to  them,  and  into  which  from  another  window  clean 
underclothing  was  passed.  We  have  described  the  arrangement  as 
used  in  the  American  Army  in  an  article  on  army  sanitation.52 

For  the  purpose  of  keeping  lice  away  from  the  body,  naphthalin 
sprinkled  through  the  underclothing  probably  has  some  effect. 

Kerosene  or  gasoline  when  applied  to  clothing  in  small  quantities 
may  keep  insects  from  lodging  in  clothing. 

Various  soaps  and  ointments  have  been  made  in  which  petroleum, 
kerosene  or  naphthalin  have  been  used  as  ingredients,  and  these 
have  been  applied  by  smearing  along  the  seams  of  the  clothing, 
under  the  arm  pits,  etc. 

An  excellent  method  for  personal  prophylaxis  has  been  the 
spraying  of  crude  creosote  oil  on  the  inner  and  outer  clothing.  This 
has  been  used  successfully  by  Pappenheimer  and  Mueller.  A  good 
way  of  killing  insects  that  may  have  wandered  into  the  clothing 
during  hospital  or  other  duties  in  the  course  of  the  day  is  to  drop 
the  clothing  into  a  dress  suit  bag  or  other  fairly  tight  container 
and  pouring  in  an  ounce  or  two  of  chloroform,  closing  it  for  the 
night.  This  will  not  always  kill  nits. 

62Zinsseiv  Military  Surgeon   November,  1918. 


956  DISEASES  CAUSED  BY   FILTRABLE  VIRUS 

Much  might  be  written  about  the  various  substances  that  can 
be  applied  to  the  skin  and  clothing  to  keep  insects  away,  but  none 
of  these  means  are  infallable  or  sufficiently  safe  to  be  relied  upon. 
The  best  possible  method,  after  all,  is  the  use  of  shower  baths  of 
hot  water,  the  plentiful  use  of  soap,  combined  with  steam  disin- 
festation  of  the  clothing,  and  clipping  of  hair  and  beard,  etc. 

Gaseous  Disinfectants  for  Rooms,  Clothing,  etc. — It  is  an  im- 
portant practical  fact  that  formaldehyd,  in  spite  of  its  powerful 
action  upon  bacteria,  is  a  weak  insecticide  and  cannot  be  relied  upon 
to  kill  lice,  mosquitoes,  or  fleas. 

Better  than  formaldehyd  is  S02  gas  used  in  quantities  of  two 
to  three  pounds  of  sulphur  per  1,000  cubic  feet,  with  the  simultaneous 
evaporation  of  water.  (Clayton  apparatus.) 

Hydrocyanic  acid  gas  is  also  very  efficient,  but  of  course  ex- 
tremely poisonous  and  dangerous  unless  used  in  a  proper  way. 

The  most  important  facts  concerning  these  gases,  their  generation 
and  application,  have  been  dealt  with  in  a  preceding  section. 

For  the  wholesale  disinfestation  of  clothing,  fomites,  etc.,  in 
connection  with  louse  infested  populations,  clothing,  blankets,  etc., 
the  experience  of  the  late  war  has  shown  that  the  most  practical 
systems  are  those  depending  upon  the  application  of  heat. 

Heat  may  be  applied  as  dry  heat  or  moist  heat. 

Our  own  experience  has  taught  us  that  the  surest  and  most 
foolproof  method  of  disinfesting  large  quantities  of  material  during 
epidemics  is  by  the  use  of  large  autoclave  drums  placed  on  trucks 
such  as  the  Foden-Thresh  autoclave  lorries,  which  consist  of  large 
autoclaves  with  steam  jackets  so  arranged  that  clothing,  etc.,  can 
be  exposed  to  steam  under  slight  pressure  (about  five  pounds),  then 
the  connection  between  the  inner  and  outer  jackets  closed  and  the 
material  dried  in  the  same  chamber  before  removal.  The  steam  is 
supplied  from  the  motor  since  such  lorries  are  usually  steam  driven. 

In  the  field,  dry  heat  chambers  can  be  constructed  consisting  of 
well  sealed  huts  within  which  small  brick  furnaces  or  tin  stoves, 
with  stovepipe  arrangements  surrounding  the  walls  are  used  for 
heating  purposes.  These  dry  heat  disinfestors  are  not  as  uniformly 
practical  or  foolproof  as  are  the  methods  in  which  steam  under 
pressure  is  applied,  but  it  may  be  necessary  to  use  them  when  other 
means  are  not  available.  Detailed  descriptions  cannot  be  given  here, 
but  a  little  ingenuity  with  attention  to  proper  size  in  relation  to 
heating  apparatus,  proper  distribution  of  heat  with  tin  pipes,  and 


TYPHUS  FEVER,  TRENCH  FEVER,  ETC.         957 

provision  for  the  circulation  of  air  by  proper  vent  holes  will  yield 
good  results.  In  the  so-called  " Canadian"  type  of  hut  the  heat  is 
applied  from  below  by  digging  a  hole  in  the  floor  of  the  hut  which 
connects  with  the  outside  through  a  small  tunnel,  in  which  a  furnace, 
constructed  in  a  variety  of  ways,  can  be  placed,  and  a  glowing  coal 
fire  maintained.  According  to  Bacot53  and  others  it  has  been  found 
that  nits  protected  by  a  single  layer  of  khaki  cloth  are  killed  in 
fifteen  minutes  at  52°  C.  The  heat  of  such  huts  must  be  carefully 
controlled,  a  matter  which  can  be  done  either  by  thermometers  or, 
as  advised  by  Bacot,  by  hanging  in  various  places  small  tubes 
containing  paraffin  or  stearin,  with  a  melting  point  of  60°  or  over. 
Excellent  methods  of  applying  the  various  forms  of  disinfestation 
by  heat  are  those  which  were  originated  by  Dr.  Richard  Strong 
in  Serbia,  in  which  disinfestation  trains  with  a  shower  bath  car, 
a  steam  sterilizing  car  made  of  a  converted  refrigerator  truck,  were 
drawn  by  an  engine,  which  supplied  the  steam  for  the  disinfestors, 
the  hot  water  for  the  baths  and  the  motor  power. 

THE    RICKETTSIA    BODIES 

In  1910  during  their  work  in  Mexico,  Ricketts  and  Wilder 
observed  small  ovoid  bacterium-like  bodies  in  the  intestinal  canals 
of  lice  which  had  fed  on  typhus  cases.  They  described  them  as 
showing  polar  staining,  with  slightly  stained  or  entirely  unstained 
centers  and  as  having  the  general  appearance  of  very  small 'bacilli. 
Similar  observations  were  made  by  Prowazek  and  by  Sergent,  Foley 
and  Vialatte  in  1913,  though  the  identity  of  the  bodies  seen  by 
these  observers  with  those  of  Ricketts  and  Wilder  was  not,  at  first 
clear.  Most  of  the  original  observations  were  made  on  typhus 
material,  but  subsequently  Wolbach  saw  similar  appearances  in  the 
endothelial  cells  and  vessel  walls  of  animals  infected  with  Rocky 
Mountain  Spotted  Fever  which  he  believed  to  be  probably  identical 
with  diplococcus-like  structures  described  in  the  blood  in  the  same 
disease  by  Ricketts  a  few  years  earlier.  Still  later  bodies  of  the 
same  general  appearance  were  noticed  in  lice  taken  from  Trench 
Fever  cases  and  in  lice  collected  from  the  bodies  of  normal  human 
beings.  The  peculiar  staining  properties,  frequently  intracellular 
position,  minute  size  and  pleomorphic  structure  of  these  peculiar 
bodies  suggested  to  many  of  these  workers  the  possibility  that  they 

63  Bacot,  Brit.  Med.  Jour.,  2,  1917,  151. 


958  DISEASES  CAUSED  BY   FILTRABLE   VIRUS 

might  represent  a  group  of  parasitic,  and  perhaps  pathogenic,  or- 
ganisms not  hitherto  observed.  Von  Prowazek  did  not  believe 
these  small  bodies  to  be  bacteria,  and  from  the  beginning  took  the 
position  that  they  were  more  likely  to  belong  to,  or  be  closely 
related  to  the  protozoa.  Da  Rocha-Lima,  who  has  studied  them 
particularly  in  their  relationship  to  typhus  fever,  gave  the  appear- 
ances which  he  saw  in  typhus  lice  the  name  of  Rickettsia  prowazeki. 
In  the  course  of  numerous  investigations  upon  the  etiological  signifi- 
cance of  these  peculiar  appearances,  especially  in  connection  with 
Typhus  fever,  Rocky  Mountain  Spotted  Fever,  and  Trench  Fever,  many 
workers  have  confirmed  the  observations  of  the  earlier  observers,  and 
while  it  is  quite  impossible  at  the  present  time  to  classify  them  with 
any  degree  of  certainty  either  with  the  bacteria  or  the  protozoa,  the 
various  forms  described  possess  sufficient  similarity  to  each  other  to 
warrant  the  establishment  of  a  tentative  group.  In  describing  them 
in  a  separate  section  we  do  not  mean  to  imply  that,  at  the  present 
time,  it  is  absolutely  certain  that  they  are  parasites.  But  this  seems 
so  likely,  and  their  etiological  relationship  to  the  diseases  mentioned 
has  been  suggested  by  so  many  careful  investigations,  that  clearness 
of  treatment  at  the  present  time  fully  justifies  such  segregation  into 
a  separate  group. 

The  appearances  which  are  classified  together  as  Rickettsia  are 
minute,  ovoid  or  bacterium-like  bodies.  They  are,  as  a  rule,  ex- 
tremely small,  the  smallest  forms  being  more  minute  than  the 
smallest  known  bacteria,  measuring  about  0.3  to  0.5  of  a  micron. 
Larger  forms  more  bacillary  in  appearance,  may  be  observed,  and 
it  is  suggested  that  the  Rickettsia  bodies  go  through  a  develop- 
mental cycle.  The  small  forms  often  appear  in  the  "diplo"  form 
and  some  German  observers  have  described  capsule-like  halos  around 
groups  of  two. 

They  are  all  very  difficult  to  stain.  The  ordinary  aniline  dyes 
stain  them  either  very  faintly  or  not  at  all.  Prolonged  staining 
with  Giemsa  gives  them  a  faint  reddish  blue  tinge.  They  do  not 
retain  the  Gram  stain. 

They  are  non-motile. 

Up  to  the  present  time  none  of  the  Rickettsia  have  been  cul- 
tivated, with  the  exception  of  one  form  observed  in  the  sheep  louse 
which  grows  aerobically  on  glucose-blood-agar. 

All  of  them  have  an  insect  host  which  acts,  in  the  case  of  the 
pathogenic  Rickettsia,  as  the  transmitting  agent, 


TYPHUS  FEVER,   TRENCH  FEVER,   ETC, 


959 


According  to  experiments  of  Rickctts  and  Wilder,  da  Rocha- 
Lima,  Sergent  and  his  co-workers,  some  of  the  Rickettsia  can  pass 
into  the  egg  of  the  louse  and  thus  be  inherited  from  one  generation 
to  the  other. 

Most  of  them,  unlike  bacteria  and  more  resembling  protozoa, 
appear  to  enter  the  cells  of  the  host  as  intracellular  parasites. 

The  sub-classification  of  the  Rickettsia  has  been  tentatively 
attempted  by  Wolbach  by  whose  courtesy  we  are  enabled  to  insert  the 
following  table : 


Insects 


Arachnida 
Acarina 


Mallophaga 
Corrodentia 


Hemiptera 


Diptera 


Siphonaptera 


Wulhaoh's  Tentative  Classification 

Melophagus  ovinuft  (sheep  "louse"  or  "tick") 

Rickettsia  melophagi    Noller 

Psocus  Sp.?  (dust  louse) 
Unnamed  rickettsia .  . . 


1917 


Sikora 1918 


Pediculus  humanus  (human  louse) 

Rickettsia  prowazeki Hegler  and  yon  Prowazek. . 

da  Rocha-Lima 

Rickettsia  (rocha-lima?) Weigl,  oral  statement 

Rickettsia  pediculi Munk  and  da  Rocha-Lima. 

Rickettsia  quintana Munk  and  da  Rocha-Lima. 

Rickettsia  wolhynica Toepfer 

Cimex  (Acanthia)  lectularius  (bed  bug) 

Rickettsia  lectularius  .  .  .  .  Bacot .  . 


1914 
1916 
1920 
1917 
1917 
1916 

1921 


Culex  pipiens  (Mosquito,  Europe) 

Unnamed  rickettsia Noller,  quoted  by  Sikora.  .  .    1920 

(  Ctenocephalus  felis  (cat  flea) 

Rickettsia  ctenocephali Sikora 1918 

Cfenopsylla  musculi  (mouse  flea) 

Unnamed  rickettsia ...  .  .  Sikora .  . 


1918 


Dermacentor  venustus  (wood  tick,  U.  S.) 
Dermacentroxenus  rickettsi .  . 


.Ricketts...  .    1909 


Leplus  (Tromibidium)  akamushi  (harvest  mite,  Japan) 
Unverified  quotation  by  Sikora 

Dermanyssu*  Sv.f      (bird  mite,  Europe) 

Unnamed,  Noller;  quoted  by  Sikora 


1920 


1920 


In  proposing  this  classification,  however,  it  should  be  said  in 
justice  to  Wolbach  that  he  introduces  it  by  stating  definitely  that 
a  reliable  classification  of  the  Rickettsia  is  impossible  at  the  present 
time,  and  that  he  believes  that  there  have  already  been  included 
under  this  heading  a  number  of  unrelated  forms.  He  states  that 
the  Rickettsia  of  the  sheep  louse  has  little  to  distinguish  it  from 
bacteria  and  that  the  Rickettsia  seen  in  connection  with  typhus 
fever  has  peculiarities  which  separate  it  from  others.  The  Rickettsia 
studied  by  him  in  connection  with  Rocky  Mountain  Spotted  Fever 
resembles  somewhat  the  Rickettsia-prowazeki  seen  in  typhus,  and 
both  of  them  are  quite  unlike  the  "morphologically  simple "  one 
observed  in  connection  with  trench  fever.  Wolbach  summarizes  his 
reasons  for  constructing  a  table  of  classification  by  saying  that  he 
believes  it  warranted  since  "they  are  forms  of  microorganisms 


000  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

primarily  adapted  to  insect  tissues,  with  occasional  representatives 
pathogenic  for  animals." 

For  more  detailed  analysis  of  the  Rickettsia,  we  refer  the  reader 
to  the  Harvey  Lecture  and  to  the  articles  on  typhus  investigations 
in  Poland  now  being  prepared  for  publication  by  Wolbach,  Todd 
and  their  associates. 

Abstracting  from  the  further  analysis  of  the  Rickettsia  sent  us 
with  this  table  by  Wolbach,  we  may  mention  the  following  points. 
The  R.  melophagia  is  not  pathogenic  and  is  the  only  one  that  has 
been  cultivated  upon  glucose-blood-agar.  The  R.  cprrodentia  of  the 
dust  louse  is,  likewise,  not  associated  with  any  mammalian  host.  It 
lives  extracellularly  in  the  stomach  of  the  louse  and  is  apparently 
non-pathogenic.  The  R.  pediculi  quintana  and  wolhynica  are  prob- 
ably identical,  according  to  Wolbach  and  Todd.  They  are  more 
uniform  in  morphology  than  the  typhus  one  and  are  easier  to  stain. 
They  occur  extracellularly  in  the  louse's  stomach,  adhere  to  the 
cuticular  epithelium,  and  may  invade  the  epithelial  cells.  They  are 
transmitted  to  the  egg.  Their  etiological  association  with  trench  fever 
has  been  suggested. 

The  R.  prowazeki  is  pleomorphic  and  seems  to  be  exclusively 
intracellular  in  the  louse.  It  seems  to  be  more  susceptible  to  drying 
and  to  heat  than  the  preceding  ones.  It  is  the  one  studied  by  da 
Rocha-Lima  and  others  connection  with  typhus  fever. 

The  R.  lectularius  of  the  bed  bug  is  non-pathogenic,  but  mor-. 
phologically  resembles  R.  prowazeki. 

The  ones  occurring  in  mosquitoes  and  in  cat  and  mouse  fleas  are 
non-pathogenic. 

The  Dermacentroxenus  rickettsi  is  the  Rickettsia  body  which  has 
been  associated  by  a  number  of  writers  with  Rocky  Mountain 
Spotted  Fever.  Wolbach  includes  it  in  the  general  classification, 
though  he  has  found  many  differences  between  it  and  the  other 
Rickettsia.  Wolbach  states  that  it  is  less  bacterium-like  than  any 
of  the  other  Rickettsia  and  many  forms  show  red  and  blue  staining 
materials.  Unlike  the  Prowazeki,  it  does  not  show  the  thread-like 
forms.  In  the  louse  he  states  that  the  Prowazeki  continues  to  mul- 
tiply in  the  gastric  epithelium  and  eventually  causes  the  death  of 
the  louse  by  interfering  with  digestion.  The  Dermacentroxenus, 
however,  after  multiplying  within  the  nucleus  chiefly,  floods  all  the 
tissues  of  the  tick  and  then  diminishes  in  numbers,  leaving  behind 
in  the  salivary  gland  and  some  other  tissues,  forms  which  Wolbach 
regards  as  a  resistant  stage. 


SECTION  V* 

THE  HIGHER  BACTERIA,  MOLDS  AND  FUNGI 


CHAPTER   XLIX 

THE  HIGHEB  BACTEKIA 

( Chlamydobacteriaceoe,  TricJiomycetes,  Microsiphonales) 

STANDING  midway  between  the  true  bacteria  and  the  more  complex 
molds,  there  are  a  number  of  pathogenic  microorganisms  which  offer 
great  difficulties  to  classification.  These  forms  resemble  the  hyphomy- 
cetes  in  the  gross  appearance  of  the  cultures,  which  are  dry,  tough, 
wrinkled  and  sometimes  covered  with  a  down  of  aerial  outgrowths. 
Morphologically  they  are  made  up  of  filaments  which  often  show  at 
the  ends  chains  of  round  bodies  analogous  to  arthrospores.  In  the  size 
and  structure  of  their  component  cells  they  are,  however,  far  more 
like  the  bacteria.  The  component  cells  of  the  filaments  are  usually 
about  0.3  micron  and  rarely  more  than  1  micron  in  diameter.  They 
frequently  stain  unevenly  but  show  no  definite  nuclei  and  the  round 
spore-like  cells  are  about  the  size  of  micrococci.  In  the  classification 
of  Migula  most  of  these  forms  have  been  placed  in  a  rather  hetero- 
geneous group,  the  Chlamydobacteriaceae.  By  other  authors,  notably 
Lachner-Sandoval,1  Berestnew,2  and  by  Petruschky,3  the  close  relation- 
ship of  these  forms  to  the  higher  hyphomycetes  has  been  emphasized 
and  they  have  been  grouped  as  a  subdivision  of  the  true  fungi  under 
the  family  name  of  Trichomycetes. 

*  For  a  careful  revision  of  this  Section  we  are   indebted  to  Dr.  J.  Gardner 
Hopkins. 

*  Lachner-Sandoval,  "Ueber  Strahlenpilze. "     Diss.  Strassburg,   1898. 
2  Berestnew,  Eef.  Cent,  f.  Bakt.,  xxiv,  1898. 

'Petruschky,  in  Kolle  und  Wassermann,  "Handbuch, "  etc. 

961 


962 


THE   HIGHER   BACTERIA,    MOLDS   AND   FUNGI 


Petruschky  proposes  the  following  clear  schematization,  which, 
even  though  possibly  defective  from  a  purely  botanical  point  of  view, 
is  at  least  serviceable  for  the  purposes  of  the  bacteriologist. 

Hyphomycetes 


True  molds 


Trichomvcetes 

r 


1 

Leptothrix 


Cladothrix 


Streptothrix 


Actinomyces 


Leptofhrix  is  used  to  designate  those  forms  which  appear  as  simple 
threads  without  branching. 

Cladothrix  is  a  thread-like  form  in  which  false  branching  may  be 
recognized.  By  false  branching  is  meant  an  appearance  resulting 
from  the  fragmentation  of  threads.  The  terminal  cell  breaks  away 
from  the  main  stem,  is  set  at  an  angle  by  the  elongation  of  the  thread 


FIG.  101. — CLADOTHRIX,  SHOWING  FALSE  BRANCHING. 

itself,  and,  as  both  continue  dividing,  the  simulation  of  true  branching 
is  produced. 

Streptofkrix  denotes  forms  with  numerous  true  branches  and  spores 
which  usually  appear  in  chains. 

Actinomyces  is  of  more  complicated  structure,  characterized  by  the 
formation  of  club-shaped  ends  and  the  stellate  arrangement  of  its 
threads. 

Concerning  the  use  of  the  last  three  generic  names  there  lias  been 
much  controversy,  which  has  recently  been  discussed  with  a  full 


THE  H1GHE11   BACTERIA 


963 


bibliography  by  Breed  and  Conn  (J.  Bacteriol.,  1919,  iv,  585).  The 
outcome  of  the  matter  seems  to  be  that  the  genus  leptothrix  may  stand 
as  representing  filamentous  forms  without  branching,  of  which  our 
knowledge  is  very  incomplete,  and  that  there  are  two  distinct  groups 
of  pathogenic  species — Nocardice,  which  are  aerobic,  and  the  actimo- 
myces,  which  are  anaerobic.  The  former,  at  least,  includes  a  large 
number  of  related  species. 


FIG.  102. — STREPTOTHRIX,  SHOWING  TRUE  BRANCHING. 


LEPTOTHRIX 

Members  of  the  leptothrix  group  have  been  observed  in  connection 
with  inflammations  of  the  mouth  and  pharynx  by  Frankel,*  Michelson,5 
Epstein,6  and  others.  In  many  of  these  cases  the  organism  was  identi- 
fied by  morphology  chiefly,  pure  cultures  not  having  been  obtained. 
The  disease  in  none  of  these  cases  was  accompanied  by  severe  systemic 
symptoms  and  it  is  likely  that  when  found  in  human  beings  the 
organisms  may  be  regarded  simply  as  comparatively  harmless  sapro- 
phytes appearing  in  connection  with  some  other  specific  inflammation. 

Cultivation  of  the  Leptothrices  is  not  easy  and  has  been  successful 
only  in  the  hands  of  Vignal7  and  Arustamoff.8 


4  Frankel,  Eulenburg's  ' '  Kealencycl.  <1.  gesam.  Heilkunde, "  1882. 

5  Michelson,  Berl.  kliu.  Woch.,  ix,  1889. 

6  Epstein,  Prag.  med.  Woch.,  1900. 

7  Vignal,  Ann.  do  phys.,  viii,  1886. 

8  Arustamoff,  Quoted  from  Petruschky,  loc.  cit. 


904  THE  HIGHER  BACTERIA,   MOLDS  AND  FUNGI 


NOCARDIA 

Streptothrix,  Cladothrix,  Oospora,  Discomyces. — This  genus  in- 
cludes a  large  group  of  aerobic  organisms  which  grow  in  branch- 
ing filaments  made  up  of  bacteria-like  units.  English  medical 
writers  more  frequently  refer  to  them  as  streptothrices,  but,  as  the 
name  Streptothrix  is  applied  to  a  group  of  common  saprophytjc  fungi 
with  coarse  filaments,  it  cannot  be  properly  used  for  these  organisms. 
Saprophytic  varieties  of  nocardia  are  numerous  and  pathogenic  strains 
have  also  been  reported  as  the  cause  of  varied  infections  in  man  and 
animals.  Nocard9  described  a  member  of  this  group  as  the  etiological 
factor  in  a  glanders-like  disease,  "farcin  du  boeuf,"  occurring  in 
Guadeloupe,  which  he  called  actinomyces  farcinica.  The  first  human 
case  was  that  of  Eppinger,10  who  cultivated  from  a  brain  abscess  an 
organism  which  he  called  Cladothrix  asteroides  on  account  of  the 
star-like  appearance  of  the  young  colonies  on  agar.  He  also  found 
the  organisms  in  sections  of  the  bronchial  lymph-nodes  and  believed 
the  invasion  had  occurred  through  the  respiratory  tract.  Since  then 
a  number  of  fatal  systemic  infections  due  to  similar  organisms  have 
been  described  by  Petruschky,11  Berestnew,12  Flexner,13  MacCallum,14 
Norris  and  Larkin15  and  others,  and  an  apparently  identical  organism 
was  isolated  by  Musgrave  and  Clegg  in  a  case  of  Madura  foot.  A 
summary  of  the  various  cases  up  to  1921  has  been  made  by  Henrici 
and  Gardner  (J.  Infect.  Dis.,  1921,  xxviii,  232).  In  most  of  these 
cases  the  portal  of  entry  was  the  respiratory  tract,  but  a  few  began 
as  wound  infections.  Strains  from  various  cases  have  been  considered 
by  some  to  be  identical,  by  others  to  represent  a  number  of  closely 
related  species. 

Morphology.  —  Morphologically  the  nocardice  show  considerable 
variation.  In  material  from  infectious  lesions  they  have  most  often 
appeared  as  rods  and  filaments  with  well-marked  branching.  Occa- 
sionally the  filaments  are  long  and  intertwined,  and  branches  have 


9  Nocard,  Ann.  de  1'inst.  Pasteur,  ii,  1888. 

10  Eppinger,  Wien.  klin.  Woch,,  1890. 

11  Petruschky,  Verhandl.  <1.  Kongr.  f.  iiniere  Mediz.,  1898. 
"Berestneff,  Zeit.  f.  Hyg.,  xxix,   1898. 

13  Flexner,  Jour.  Exp.  Med.,  iii,  1896. 

14  MacCallum,  W.  E.,  Centralbl.  f.  Bakt.,  I,  O,  1902,  xxxi,  529. 

15  Norris  and  Larkin,  Proc.  of  N.  Y.  Path.  Soc.,  March,  1899. 


THE  HIGHER  BACTERIA  965 

shown  bulbous  or  club-shaped  ends.  In  Norris  and  Larkin's  case, 
the  young  cultures  in  the  first  generations  seem  to  have  consisted 
chiefly  of  rod-shaped  forms  not  unlike  bacilli  of  the  diphtheria  group, 
showing  marked  metachromatism  when  stained  with  Loeffler's 
methylene-blue.  They  are  easily  stained  with  this  dye  or  with 
aqueous  fuchsin.  Many  strains  are  acid-fast,  but  decolorize  some- 
what more  readily  than  do  tubercle  bacilli.  In  tissue  sections  they 
may  be  demonstrated  by  the  Gram-Weigert  method. 

Cultivation. — The  organism  develops  slowly  on  ordinary  agar 
or  gelatin  plates,  forming  visible  colonies  in  from  two  to  five  days. 
Later  it  forms  a  membrane  somewhat  adherent  to  the  surface  which 
soon  becomes  wrinkled.  It  is  at  first  white  but  later  turns  yellow 
or  even  a  brilliant  orange.  On  broth  they  grow  as  a  thick  pellicle 
or,  occasionally  as  a  flocculent  precipitate.  Most  strains  have  not 
liquefied  gelatin  or  altered  litmus  milk,  but  liquefying  strains  have 
been  described.  All  strains  have  proved  highly  virulent  for  guinea- 
pigs  and  somewhat  less  so  for  rabbits,  producing  in  the  animals 
lesions  indistinguishable  from  tuberculosis. 

NOCARDLE  IN  RAT-BITE  FEVER. — In  the  cases  of  fatal  septicemia 
following  rat  bites,  Schottmueller16  and  Blake17  have  recovered 
nocardias  from  the  blood.  It  has  since  been  shown,  however,  that  this 
disease  is  due  to  infection  by  treponemata. 

Streptothrix  of  Rosenbach. — A  species  of  nocardia  undoubtedly 
different  from  the  asteroides  group  has  been  described  by  Rosen- 
bach18  as  the  cause  of  an  indolent  dermatitis  of  the  fingers  and  toes 
known  as  erysipeloid. 

ACTINOMYCES 

(Streptothrix  Israeli,  Kruse;  Discomyces  bovis,  Brumpt ;  Colmistrep- 
tofkrix  Israeli,  Pinoy) 

Among  the  diseases  caused  by  the  Trichomycetes  or  higher  bac- 
teria, the  most  important  is  actinomycosis.  Occurring  chiefly  in 
some  of  the  domestic  animals,  notably  in  cattle,  the  disease  is 
observed  in  man  with  sufficient  frequency  to  make  it  of  great  clinical 
importance.  In  cattle  the  specific  microorganism  which  gives  rise 

"Schottmueller,   Dermat.  Wchnschrft.,   1914,   LVIII,   Sup.   77. 
"Blake,  F.  G.,  Jour.  Exp.  Med.,  1916,  XXIII,  39. 
18  Rosenbach,  Arch.  f.  klin.  Chirurg.  1887,  xxiv,  346. 


966 


THE   HIGHER  BACTERIA,    MOLDS  AND   FUNGI 


to  the  disease  was  first  observed  by  Bellinger19  in  1877.  In  the 
following  year  Israel20  discovered  a  similar  microorganism  in  human 
cases. 

The  parasites  appear  in  the  pus  from  discharging  lesions  as  small 
granular  bodies,  plainly  visible  to  the  naked  eye  and  somewhat 
resembling  sulphur  granules,  of  a  grayish  or  of  a  pale  yellow  color. 
In  size  they  measure  usually  a  frac- 
tion of  a  millimeter.  Ordinarily  they 
are  soft  and  easily  crushed  under  a 
cover-slip,  but  occasionally,  especially  in 
old  lesions,  they  may  be  quite  hard, 
owing  to  calcification. 


FIG.  103. — ACTINOMYCES  GRANULE  CRUSHED 
BENEATH  A  COVER-GLASS.  Unstained. 
Low  power.  Shows  radial  striations.  (After 
Wright  and  Brown.) 


FIG.  104. — ACTINOMYCES 
GRANULE  CRUSHED  BE- 
NEATH A  COVER-GLASS. 
Unstained.  The  prepara- 
tion shows  the  margin  of  the 
granule  and  the  "clubs." 
(After  Wright  and  Brown) . 


Microscopically  they  are  most  easily  recognized  in  fresh  prepara- 
tions prepared  by  crushing  the  granules  upon  the  slide  under  a 
cover-slip  and  examining  them  without  staining.  They  may  be 
rendered  more  clearly  visible  by  the  addition  of  a  drop  or  two  of 
20  per  cent  potassium  hydrate.  When  the  granules  are  calcareous, 
the  addition  of  a  drop  of  concentrated  acetic  acid  will  facilitate 


™  Bollinger,  Deutsch.  Zeit.  f.  Thiermed.,  iii,  1877. 

20  Israel,  Virch.  Arch.,  74,  1878  and  1879,  LXXVIII,  421. 


THE   HIGHER   BACTERIA  967 

examination.  Fresh  preparations  may  be  examined  after  staining 
with  Gram's  stain.  Observed  under  the  microscope,  the  granules 
appear  as  rosette-like  masses,  the  centers  of  which  are  quite  opaque 
and  dense,  appearing  to  be  made  up  of  a  closely  meshed  network 
of  filaments.  Around  the  margins  there  are  found  radially  arranged 
striations  which  in  many  cases  end  in  characteristically  club-shaped 
bodies.  Inside  of  the  central  network  there  are  often  seen  coccoid 
or  spore-like  bodies  which  have  been  variously  interpreted  as  spores, 
as  degeneration  products,  and  as  separate,  cocci  fortuitously  found 
in  symbiosis  with  the  actinomyces.  Individually  considered,  the 
central  filaments  have  approximately  the  thickness  of  an  anthrax 
bacillus  and  are,  according  to  Babes,21  composed  of  a  sheath  within 


FIG.  105. — BRANCHING  FILAMENTS  OF  ACTINOMYCES.     (After  Wright  and  Brown.) 

which  the  protoplasm  contains  numerous  and  different  sized 
granules. 

About  the  periphery  of  the  granules  the  free  ends  of  the  filaments 
become  gradually  thickened  to  form  the  so-called  actinomycosis 
' '  clubs. ' '  These  clubs,  according  to  most  observers,  must  be  regarded 
as  hyaline  thickenings  of  the  sheaths  of  the  threads  and  are  believed 
to  represent  a  form  of  degeneration  and  not,  as  some  of  the  earlier 
observers  believed,  organs  of  reproduction.  They  are  homogeneous, 
and  in  the  smaller  and  presumably  younger  granules  are  extremely 
fragile  and  soluble  in  water.  In  older  lesions,  especially  in  those 
of  cattle,  the  clubs  are  more  resistant  and  less  easily  destroyed. 

They  appear  only  in, the  parasites  taken  from  active  lesions  in 
animals  or  man,  or,  as  Wright22  has  found,  from,  cultures  to  which 


21  Bales,  Yirch.  Arch.,  105,  1886. 

22  J.  H.   Wright,  Jour.  Med.  Res.,  1905,  vii,  349. 


968  THE  HIGHER  BACTERIA,   MOLDS  AND  FUNGI 

animal  serum  or  whole  blood  has  been  added.  In  cultures  from 
media  to  which  no  animal  fluids  have  been  added,  such  as  glucose 
agar  or  gelatin,  no  clubs  are  found.  In  preparations  stained  by 
Gram's  method  the  clubs  give  up  the  gentian-violet  and  take  counter- 
stains,  such  as  eosin. 

The  coccus-like  bodies  found  Occasionally  lying  between  the  fila- 
ments of  the  central  mass,  most  observers  now  agree,  do  not 
represent  anything  comparable  to  the  spores  of  the  true  hyphomy- 
cetes.  In  many  cases  they  are  unquestionably  contaminating  cocci ; 
in  others  again  they  may  represent  the  results  of  degeneration  of 
the  threads. 

In  tissue  sections,  the  microorganisms  may  be  demonstrated  by 
Gram's  method  of  staining  or  by  a  special  method  devised  by 
Mallory.23  This  is  as  follows  for  paraffin  sections : 

1.  Stain  in  saturated  aqueous  eosin  ten  minutes. 

2.  Wash  in  water. 

3.  Anilin  gentian-violet,  five  minutes. 

4.  Wash  with  normal  salt  solution. 

5.  Gram's  iodin  solution  one  minute. 

6.  Wash  in  water  and  blot. 

7.  Cover  with  anilin  oil  until  section  is  clear. 

8.  Xylol,  several  changes. 

9.  Mount  in  balsam. 

Cultivation. — The  isolation  of  actinomyces  from  lesions  may  be 
easy  or  difficult  according  to  whether  the  pus  is  free  from  con- 
tamination or  whether  it  contains  large  numbers  of  other  bacteria. 
In  the  latter  case  it  may  be  almost  impossible  to  obtain  cultures. 
The  descriptions  of  methods  of  isolation  and  of  cultural  character- 
istics given  by  various  writers  have  shown  considerable  differences. 
The  most  extensive  cultural  work  has  been  done  by  Bostroem,24 
Wolff  and  Israel,  and  by  J.  H.  Wright,  Bostroem  has  described  his 
cultures  as  aerobic,  but  Wolff  and  Israel25  and  Wright26  agree  in 
finding  that  the  microorganisms  isolated  by  them  from  actinomycotic 
lesions  grow  but  sparsely  under  aerobic  conditions  and  favor  an 


:3  Mallory,  Method   No.    1,  Mallory  and   Wright,  "Path.    Technique/'   Phila. 
1908. 

24  Bostroem,  Beitr.  z.  path.  Anat.  u.  z.  allg.  Path.,  ix,  1890. 

25  Wolff  und  Israel,  Virch.  Arch.,  1891,  cxxvi,  4. 
M  J.  H.  Wright,  Jour.  Mod.  Res.,  viii,  1905. 


THE   HIGHER   BACTERIA  969 

environment  which  is  entirely  free  from  oxygen,  or  at  least  contains 
it  only  in  small  quantities.  The  method  for  isolation  recommended 
by  Wright  is,  briefly,  as  follows:  Pus  is  obtained,  if  possible,  from 
a  closed  lesion  and  washed  in  sterile  water  or  broth.  The  granules 
are  then  crushed  between  two  sterile  slides  and  examined  for  the 
presence  of  filaments.  If  these  are  present  in  reasonable  abundance, 
the  material  is  distributed  in  tubes  of  glucose  agar,  which  are  then 
allowed  to  solidify.  If  these  first  cultivations  show  a  large  number 
of  contaminations,  Wright  recommends  the  preservation  of  other 
washed  granules  in  test  tubes  for  several  weeks, -in  the  hope  that 
contaminating  microorganisms  may  thus  be  killed  by  drying  before 
the  actinomyces  lose  their  viability. 

If  cultivation  is  successful  colonies  will  appear,  after  two  to  four 
days  at  37.5°  C.,  as  minute  white  specks,  which,  in  Wright's  cultures, 
appeared  most  abundantly  within  a  zone  situated  5  to  10  millimeters 
below  the  surface  of  the  medium.  Above  and  below  this  zone  they 
are  less  numerous,  indicating  that  a  small  amount  of  oxygen 
furnishes  the  best  cultural  environment.  Upon  the  surface  of  agar 
slants,  growth,  if  it  takes  place  at  all,  is  not  luxuriant. 

In  alkaline  meat-infusion  broth  growth  takes  place  in  the  form 
of  heavy,  flocculent  masses  which  appear  at  the  bottom  of  the  tubes. 
Surface  growth  and  clouding  do  not  take  place. 

Milk  and  potato  have  been  used  as  culture  media  but  are  not 
particularly  favorable. 

Pathogenicity. — As  stated  above,  actinomycosis  occurs  spon- 
taneously most  frequently  among  cattle  and  human  beings.  It  may 
also  occur  in  sheep,  dogs,  cats,  and  horses.  Its  locations  of  pre- 
dilection are  the  various  parts  adjacent  to  the  mouth  and  pharynx. 
It  occurs  also,  however,  in  the  lungs,  in  the  intestinal  canal,  and 
upon  the  skin.  When  occurring  in  its  most  frequent  location,  the 
lower  jaw,  the  disease  presents,  at  first,  a  hard  nodular  swelling 
which  later  becomes  soft  because  of  central  necrosis.  It  often  in- 
volves the  bone,  causing  a  rarefying  osteitis.  As  the  swellings  break 
down,  sinuses  are  formed  from  which  the  granular  pus  is  discharged. 
The  neighboring  lymph  nodes  show  painless,  hard  swellings.  His- 
tologically,  about  the  filamentous  knobs  or  granules,  there  is  a 
formation  of  epithelioid  cells  and  a  small  round-cell  infiltration.  In 
older  cases  there  may  be  an  encapsulation  in  connective  tissue  and 
a  calcification  of  the  necrotic  masses,  leading  to  spontaneous  cure. 
As  a  rule,  this  process  is  extremely  chronic.  Infection  in  the  lungs 


970  THE  HIGHER  BACTERIA,   MOLDS  AND  FUNGI 

or  in  the  intra-abdominal  organs  is,  of  course,  far  more  serious. 
When  death  occurs  acutely,  it  is  often  due  to  secondary  infection. 
The  disease  is  acquired  probably  by  the  agency  of  hay,  straw,  and 
grain. 

Berestnew27  has  succeeded  in  isolating  actinomyces  from  straw 
and  hay  which  he  covered  with  sterile  water  in  a  potato  jar  and 
placed  in  the  incubator.  After  a  few  days  small  white  specks 
looking  like  chalk  powder  appeared  upon  the  stalks  which  upon 
.further  cultivation  developed  a  growth  which  he  considered  identical 
with  the  pathogenic  species  of  the  Bostroem  type.  Typical  anaerobic 
actinomyces  have  never  been  isolated  except  from  cases  of  the 
disease. 

Animal  inoculation  has  given  conflicting  results.  Bostroem  with 
his  aerobic  cultures  was  unable  to  produce  lesions.  Israel  and  Wolff 
did,  on  the  other  hand,  produce  nodules  resembling  those  seen  in 
spontaneous  infections  with  their  anaerobic  cultures,  but  the  lesions 
were  not  progressive  and  healed  spontaneously.  Wright  could 
produce  lesions  with  anaerobic  but  never  with  aerobic  strains.  He 
concluded  that  the  anaerobic  organism  was  the  true  cause  of  ac- 
tinomyces, and  that  Bostroem  was  probably  dealing  with  a  con- 
tamination. On  the  other  hand  Pinoy,  Castellani,  Brumpt,  and 
others  reviewing  the  subject  state  that  the  disease  actinomycosis,  though 
usually  produced  by  the  anaerobic  organism  is  in  some  cases  caused 
by  aerobic  organisms  belonging  to  the  genus  nocardia.  Others  have 
attempted  to  distinguish  between  strains  by  the  presence  or  absence 
of  clubs  in  the  infected  tissue,  reserving  the  name  actinomyces  for 
those  parasites  which  produce  clubs  and  calling  all  others  strepto- 
thrices  or  nocardice.  This  is  an  unsatisfactory  criterion,  however,  as 
MacCallum  was  able  to  produce  clubs  by  intraperitoneal  injection  of 
his  strain  which,  in  every  other  respect,  was  a  typical  nocardia 
asteroides. 

B.  actinomycetum  comitans. — As  in  other  mycoses  the  isolation 
of  the  parasite  in  this  disease  is  made  difficult  by  the  presence  in 
the  lesions  of  numerous  bacteria  which  overgrow  primary  cultures. 
The  contaminants  are  frequently  pyogenic  cocci  and  saprophytic 
intestinal  bacilli.  One  unusual  type  of  organism  has,  however,  been 
found  in  these  lesions  with  sufficient  frequency  to  deserve  mention. 
Wolf  and  Israel28  mention  the  presence  in  the  granules  of  numerous 

27  Berestnew,  Bef.  Cent.  Bakt.,  24,  1898. 

28  Wolf,  M.,  and  Israel,  J.,  loc.  cit. 


THE   HIGHER  BACTERIA  971 

pleomorphic  bodies  resembling  micrococci.  Similar  organisms  have 
been  noted  by  Klingler-9  and  recently  in  twenty-four  cases  by  Cole- 
brook"0  011  whose  paper  the  following  description  is  based.  In  the 
granules  they  appear  as  minute  Gram-negative  cocco-bacilli  fused 
together  into  sheets.  In  culture  they  grow  aerobically  or  anaerob- 
ically  as  minute  coherent  colonies  which  on  agar  are  smooth  and 
glistening,  in  broth,  starlike  and  frequently  adherent  to  the  sides 
of  the  tubes.  They  grow  on  simple  media  but  quickly  die  out  in 
culture.  They  form  acid  but  no  gas  on  sugars.  Guinea-pigs  and 
rabbits  may  be  killed  by  large  injections  but  no  lesions  resembling 
actinomycosis  are  produced.  It  is  doubtful  if  these  bacteria  play 
an  important  role  in  spontaneous  cases  of  the  disease. 

Actinobacillosis. — A  disease  of  cattle  simulating  actinomycosis, 
in  which  granules  with  rays  occur  in  the  exudates  has  been  described 
in  the  Argentine  by  Ligniercs  and  Spitz  and  in  England  by  Griffith.31 
Definite  filaments  were  not  found  in  the  lesions  and  cultures  yielded 
a  Gram-negative  strepto-bacillus.  One  human  case  has  been  reported 
by  Ravaut  and  Pinoy.32 


MYCETOMA    (MADURA    FOOT) 

The  disease  known  by  this  name  is  not  unlike  actinomycosis.  It 
is  more  or  less  strictly  limited  to  warmer  climates  and  was  first 
recognized  as  a  clinical  entity,  in  India,  by  Carter.33  Clinically  it 
consists  of  a  chronic  productive  inflammation  most  frequently  attack- 
ing the  foot,  less  often  the  hand,  very  infrequently  other  parts  of 
the  body.  Nodular  swellings  occur,  which  break  down  in  their 
centers,  leading  to  the  formation  of  abscesses,  later  of  sinuses.  Often 
the  bones  are  involved  and  a  progressive  rarefying  osteitis  results. 
From  the  sinuses  a  purulent  fluid  exudes,  in  which  are  found  char- 
acteristic granular  bodies.  These  may  be  hard,  brittle,  and  black, 
resembling  grains  of  gunpowder,  or  may  be  grayish-white  or  yellow 
and  soft  and  grumous.  According  to  the  appearance  of  these  granules 
different  varieties  of  the  disease  have  been  described  as  mycetoma 


*>  Klingler,  E.,  Centr.  f.  Bact.,  I,  O;  1912,  LXIL,   191. 
»  Colebrook,  L.,  Brit.  Jour.  Exp.  Path.,  1920,  I,  197. 
31  Griffith,  F.,  Jour,  of  Hyg.,  1916,  XV,  195. 
*-Ravcmt  and  Pinoy,  Presse  Med.,  1911,  XIX,  49. 
33  Carter  on  Mycetoma,  etc.,  London,  1874. 


972  THE  HIGHER  BACTERIA,   MOLDS  AND  FUNGI 

with  black,  white,  yellow,  or  red  granules.  These  different  varieties 
of  the  disease  though  clinically  similar  may  apparently  be  produced 
by  a  large  number  of  diffeernt  parasites  all  belonging  to  the  fungi 
or  higher  bacteria.  We  will  mention  below  the  few  cases  from 
which  aspergilli  and  allied  molds  have  been  isolated.  In  the  ma- 
jority of  cases,  organisms  similar  to  the  nocardia  have  been  found. 
Brurnpt  divides  them  into  two  genera:  the  madurella  having  septate 
mycelia  and  the  discomyces  which  is  without  septa,  but  this  distinc- 
tion is  not  generally  recognized.  For  a  discussion  of  the  character- 
istics of  the  various  strains  one  should  refer  to  the  works  of  Brumpt 
and  of  Castellani. 

The  parasite  of  the  commoner  black  variety  which  certainly  seems 
to  be  a  distinct  disease  has  been  carefully  studied  by  Wright  from 
whose  description  the  following  points  are  taken. 

The  small,  brittle  granules  observed  under  the  microscope  show 
a  dark,  almost  opaque  center  along  the  edges  of  which,  filaments, 
or  hyphse,  may  be  seen  in  a  thickly  matted  mass.  By  crushing  the 
granules  under  a  cover-slip  in  a  drop  of  sodium  hypochlorite  or  of 
strong  sodium  hydrate,  the  black  amorphous  pigment  is  dissolved 
and  the  structural  elements  of  the  fungus  may  be  observed.  They 
seem  to  be  composed  of  a  dense  meshwork  of  mycelial  threads  which 
are  thick  and  often  swollen,  and  show  many  branches.  Transverse 
partitions  are  placed  at  short  distances  and  the  individual  filaments 
may  be  very  long.  Spores  were  not  observed  by  Wright.  In  a 
series  of  over  fifty  cultivations  on  artificial  media  from  the  original 
lesion,  Wright  obtained  growth  in  a  large  percentage. 

In  broth,  he  obtained  at  first  a  rapid  growth  of  long  hyphae  which 
eventually  formed  a  structure  which  he  compares  in  appearance  to 
a  powder-puff. 

On  agar,  growth  appeared  within  less  than  a  week  and  spread  over 
the  surface  of  the  medium  as  a  thick  meshwork  of  spreading  hyphae 
of  a  grayish  color.  In  old  cultures  black  granules  appeared  among 
the  mycelial  meshes. 

On  potato,  he  observed  a  dense  velvety  membrane,  centrally  of  a 
pale  brown,  white  at  the  periphery.  Small  brown  droplets  appeared 
on  the  growth  in  old  cultures. 

Animal  inoculation  with  this  microorganism  has  so  far  been  un- 
successful. 


CHAPTER    L 
THE  PATHOGENIC  FUNGI1 

THE  earliest  demonstrations  of  microorganisms  as  the  causes  of 
disease  were  the  discovery  of  a  fungus  in  the  scutula  of  favus  by 
Schocnlein  in  1839  and  Langenbeck's  discovery  of  the  thrush  para- 
site in  the  same  year.  Later  the  work  of  Pasteur  and  his  followers 
showed  the  far  greater  importance  of  bacteria  as  disease-producers 
and  the  study  of  these  simpler  forms  has  since  been  given  greater 
attention.  We  must,  however,  briefly  consider  a  group  of  diseases 
known  as  the  mycoses  which  are  due  to  infection  by  the  fungi. 

In  the  broader  sense  the  term  fungi  is  used  to  include  all 
thallophyta  (plants  without  stems,  roots,  or  leaves)  which  are  devoid 
of  chlorophyl  or  its  analogues,  and  which  consequently  are  limited 
to  a  saprophytic  or  parasitic  existence.  In  this  sense  the  fungi  form 
a  class  of  which  the  bacteria,  or  fungi  which  reproduce  by  simple 
fission,  are  the  simplest  types.  In  the  narrower  sense  the  term  is 
applied  only  to  forms  which  reproduce  by  means  of  spores.  The 
cells  of  these  latter  organisms  are  somewhat  larger  than  those  of 
the  bacteria  and  are  usually  enclosed  in  a  well  differentiated  mem- 
brane. They  contain,  as  a  rule,  a  demonstrable  nucleus,  granules 
of  various  types  and  often  vacuoles.  Some  fungi  are  unicellular, 
as  are  the  bacteria,  but  most  are  made  up  of  many  cells  which  are 
interdependent  and  show  some  differentiation  in  form  and  function. 

Typical  fungi  are  made  up  of  cylindrical  cells,  joined  into  fila- 
ments, from  which  smaller  rounded  cells  called  spores  are  developed. 
From  these  two  elements,  filaments  and  spores,  the  fungi  build  up 
a  structure  that  differs  immensely  in  complexity  in  the  different 
species.  The  unicellular  types  such  as  the  common  yeasts  grow  in 
easily  dissociated  masses  like  bacteria  and  each  cell  combines  the 
functions  of  absorbing  food-stuffs,  of  building  them  up  into  its  own 
substance  and  of  reproducing  new  individuals.  In  the  molds,  which 
represent  simpler  multicellular  fungi,  the  filaments  lie  distinct  in 

1  This  chapter  has  been  rewritten  for  us  by  Dr.  J.  G.  Hopkins. 

973 


974  THE  HIGHER  BACTERIA,   MOLDS  AND  FUNGI 

a  loose  meshwork,  without  definite  arrangement  except  that  certain 
of  them  are  thrust  up  vertically  and  develop  spores.  In  the  higher 
types,  of  which  the  mushrooms  are  familiar  examples,  certain  fila- 
ments form  a  cobweb-like  net-work  which  is  spread  through  the 
soil  on  which  they  grow.  These  serve  to  absorb  and  pass  on  nourish- 
ment. They  connect  with  other  filaments  which  are  welded  together 
to  form  the  firm  umbrella-like  structure  which  projects  above  the 
ground.  This  is  covered  with  a  tough  protective  membrane  also 
made  up  of  closely  cohering  filaments.  Along  the  gills  on  the  under 
side  of  the  cap  are  rows  of  characteristic  cells  (basidia)  on  which 
the  spores  are  born.  These  different  portions  are  analogous  in 
function  to  the  roots,  stems,  bark,  fruit  and  seeds  of  higher  plants. 

The  gross  appearance,  the  microscopic  structure  and  especially 
the  type  of  spores  produced  are  relied  on  for  the  identification  and 
classification  of  the  various  species  of  fungi.  Consequently,  it  will 
be  necessary  to  define  some  of  the  morphological  terms  used  before 
proceeding  to  a  description  of  the  different  pathogenic  types. 

MORPHOLOGICAL    DEFINITIONS 

The  Thallus. — The  entire  vegetative  portion  of  a  fungus  is  called  the 
thallus;  the  individual  filaments  of  which  it  is  composed,  hyphae.  When  the 
hyphae  lie  in  a  loose  meshwork  without  definite  arrangement,  the  mass  is 
termed  a  mycelium  and  sometimes  this  term  is  applied  to  the  entire  thallus, 
even  when  it  develops  a  characteristic  morphology.  In  some  species  the 
hyphae  are  continuous  tubes  with  multiple  nuclei;  in  others  they  are  divided 
by  septa  into  chains  of  cylindrical  cells. 

Hyphae  which  have  special  functions  are  often  differentiated  from  the 
rest  of  the  mycelium.  In  most  of  the  pathogenic  fungi  the  thallus  is  rudi- 
mentary and  it  is  not  necessary  to  discuss  here  the  elaborate  and  somewhat 
confused  terminology  used  in  'describing  the  more  complex  forms.  In  the 
species  to  be  considered  only  the  fertile  hyphae,  i.e.,  those  that  give  rise 
to  spores  are  differentiated.  They  are  called  sporophores  or  conidiopJiores, 
these  terms  being  applied  sometimes  to  one  specialized  cell,  sometimes  to  a 
multicellular  or  branched  filament  or  to  a  bundle  of  filaments.  A  small  cell, 
or  even  a  conical  process  from  a  cell,  which  serves  as  a  point  of  attachment 
for  spores  is  called  a  sterigma;  flask-shaped  cells  of  this  type  are  called 
phialides. 

Spores. — The  term  spore  in  the  stricter  sense  means  a  rounded  reproduc- 
tive cell  analogous  in  function  to  the  multicellular  seed  of  a  higher  plant. 
As  a  rule,  a  spore  differs  in  form  from  the  parent  cell,  and  does  not  divide 
until  it  becomes  separated  from  the  thallus,  then  after  a  latent  period  it 


THE  PATHOGENIC   FUNGI  975 

germinates  and  produces  a  new  thallus.  This  meaning  is  quite  different 
from  that  in  which  the  word  is  used  when  describing  bacteria.  The  same 
term  is,  however,  often  loosely  used  for  any  rounded  cell  of  a  fungus,  whether 
it  be  a  part  of  the  body  of  the  organism,  or  an  encysted  resting  form,  or 
a  true  reproductive  type.  In  fact  the  function  of  a  cell  in  these  rudimentary 
plants  is  often  hard  to  define,  as  one. which  seems  at  first  to  be  merely  a 
component  unit  of  the  thallus  may  if  it  becomes  detached  reproduce  an  entire 
organism.  We  may,  however,  divide  all  these  rounded  cells  into  two  classes: 
(a.)  True  spores,  the  sole  function  of  which  is  reproduction;  and  (b)  Vegeta- 
tive spores,  remembering  that  the  latter  may  also  serve  to  reproduce  the 
species. 

Spores  differ  in  their  mode  of  origin  and  in  their  arrangement  on  or 
within  the  thallus.  Almost  every  species  of  fungus  produces  several  types. 
There  are  two  groups  of  reproductive  spores,  one  sexually  produced,  the  other 
asexually.  Of  the  many  names  given  to  different  forms  we  will  attempt  to 
define  only  those  which  it  will  be  necessary  to  use  in  this  chapter.  It  will 
be  found  that  many  of  these  are  used  in  a  somewhat  varying  sense  by 
different  writers. 

REPRODUCTIVE  SPORES. — An  Oospore  is  a  sexual  type  produced  by  the 
fertilization  of  a  female  cell  by  a  differentiated  male  cell. 

A  Zygospore  is  a  sexual  type  produced  by  the  fusion  of  two  undifferen- 
tiated  cells. 

Conidium  is  a  general  term  applied  to  all  asexual  reproductive  spores. 
The  term  is  by  some  writers  restricted  to  exospores  or  those  formed  by  a 
bud  which  protrudes,  from  the  membrane  of  the  parent  cell. 

Endospore  is  a  general  term  applied  to  any  spore  formed  within  the 
membrane  of  the  parent  cell. 

Ascospores  are  a  special  class  of  endospores  which  are  formed  in  a 
membrane  known  as  the  ascus,  the  number  of  spores  in  the  sack  being  limited 
to  two,  four,  or  eight,  and  constant  for  the  particular  species  producing 
them.  The  parent  cell  from  which  ascospores  are  produced  has  originally 
two  nuclei  which  fuse  into  one  before  again  dividing  to  form  the  ascospores. 
This  fusion  is  regarded  by  Dangeard  as  a  rudimentary  sexual  process.  It 
has  also  been  shown  by  Harper  and  others  that  in  certain  species  the  parent 
cell  is  the  result  of  the  fertilization  of  a  female  cell  by  a  differentiated  male 
cell.  This  is  not  true  of  some  of  the  simpler  types  such  as  yeasts. 

Basidiospores  are  exospores  produced  on  a  special  type  of  sporophore, 
known  as  a  basidium.  The  number  of  spores  on  a  basidium  is  limited  and 
constant  for  a  given  species.  Like  the  ascus,  the  basidium  has  in  certain 
species  been  found  to  have  two  nuclei  which  fuse  before  the  spores  are 
produced. 

VEGETATIVE  SPORES. — Thallospore  is  a  general  term  applied  to  cells 
morphologically  resembling  the  above  types  which  are  essentially  a  part  of 
the  vegetative  portion  of  the  fungus, 


976  THE  HIGHER  BACTERIA,   MOLDS  AND  FUNGI 

Blastospores  are  thallospores  which  develop  by  budding  from  the  end  or 
side  of  the  parent  cell  and  which  may  in  turn  throw  out  another  bud  or 
a  mycelial  filament  without  becoming  detached,  and  without  any  period  of 
latency.  The  buds  of  yeast  cells  are  familiar  examples. 

Arthrospores  are  thallospores  formed  by  the  segmentation  of  a  hypha 
into  a  chain  of  cells  at  first  cubical  and  later  rounded. 

Chlamydospores  are  single  thalospores  formed  by  the  concentration  of 
the  protoplasm  of  a  hypha  into  a  swollen  portion  of  the  filament,  the 
membrane  of  which  becomes  thickened.  They  are  purely  resting  spores  and 
are  closely  analogous  in  function  to  the  spores  of  bacteria.  They  appear 
as  roughly  spherical  thick-walled  cells  much  greater  in  diameter  than  the 
hypha3  and  are  called  intercalary  if  they  develop  in  the  course  of  a  continuous 
filament ;  terminal,  if  at  the  end  of  a  long  hypha  or  a  short  lateral  branch. 

CLASSIFICATION 

The  whole  subject  of  the  classification  of  the  fungi  is  in  confu- 
sion and  the  phylo genetic  relationship  of  the  various  groups  is 
obscure.  Even  the  identification  of  a  species  is  often  difficult,  partly 
because  some  simpler  types  show  little  that  is  distinctive  in  their 
structure  but  chiefly  because  their  morphology  varies  greatly  with 
changed  environment.  For  example,  some  of  the  blastomyces  which 
grow  in  the  animal  body  as  round  cells  reproducing  by  means  of 
blastospores,  when  placed  on  artificial  media  develop  a  mycelium 
and  conidia.  Many  of  the  higher  forms,  too,  which  are  parasitic 
on  plants  assume  on  different  hosts  forms  which  bear  no  resemblance 
to  each  other.  This  pleomorphism  has  made  the  study  of  the  fungi 
a  difficult  field.  A  species  may  be  observed  for  years  before  it 
exhibits  characteristics  which  show  clearly  its  relationship  to  certain 
other  species. 

The  modes  of  spore  production  are  the  chief  characteristics  accord- 
ing to  which  the  fungi  are  grouped — especially  those  modes  which 
seem  to  represent  a  sexual  process.  The  various  species  are  classed 
as  those  which  form  oospores,  or  ascospores,  etc.  Many,  however, 
produce  only  conidia  and  it  is  generally  considered  that  they  are 
degenerate  types  which  have  lost  the  power  of  even  rudimentary 
sexual  reproduction.  Such  varieties  are  often  grouped  together  as  the 
fungi  imperfecti.  As  to  relationships  among  these  organisms,  there 
is  little  agreement  and  almost  every  writer  has  brought  forward  a 
new  grouping  and  a  new  classification.  There  are,  however,  certain 
large  groups  which  are  generally  recognized,  the  main  characteristics 
of  which  are  shown  in  the  following  table : 


«J  'o 

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THE 

PATHOGENIC 

FUNGI 

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S3 
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s 

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o 
S 

1 

mold.  Occasionally 
in  human  lesions. 

ie  Yeasts  and  common 

| 

^ 

3n 

^2 

o 

"S 

c8 
o> 
2 
o 
02 

^ 

3 
1 

«H 

O 
0 

I 

^ 
fl 

C3 

cetes  and  dermatoph; 
this  group. 

1 

CO 

_^H 

^ 

F-H 

s 

1 

8 

a 

aj 

«r 

£> 

«H 

V 

^« 

o 

03 

o 

3 

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CO 

3 

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. 

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CC 

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£ 

PH 

A 

M 

§H 

03 

O 

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O 

S 

O 

•S 

*  V 

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03 

3 

;2 

977 


M 


ese  Most  of  the  fung 
s.  man  included. 


^  a 


•  — 

r 


g  2 


«   a 
c 


5   §^ 

L-1     M 


I. 


Jl 

II 


978  THE  HIGHER  BACTERIA,   MOLDS   AND   FUNGI 

The  Phycomycetes,  Ascomycctes  and  Hyphomycetcs  arc  the  only 
groups  that  need  be  considered  here.  The  two  former  concern  us 
chiefly  because  they  are  found  so  frequently  as  contaminants  in 
bacterial  cultures  and  will  be  briefly  discussed. 


PHYCOMYCETES 

Members  of  the  genus  Mucor  belonging  to  this  order  frequently 
appear  in  agar  plates  which  have  been  opened.  They  develop  as  a 
mesh  of  delicate  white  filaments  completely  filling  the  plate  and  press- 


FIG.  106. — MUCOR  MUCEDO.     Single-celled  mycelium  with  three  hyphse  and  one 
developed  sporangium.     (After  Kny,  from  Tnvel.) 

ing  against  the  cover.  The  sporangia  can  be  seen  with  the  naked 
eye  as  black  dots  scattered  through  the  growth.  On  opening  such  a 
plate  the  meshwork  quickly  collapses  and  forms  a  white  speckled 
feltwork  over  the  medium. 

Microscopically  the  mycelium  consists  of  branching  tubular  fila- 
ments with  or  without  septa.  The  usual  mode  of  reproduction  is  by 
asexual  spores.  These  develop  in  the  tip  of  a  hypha  which  enlarges 
to  form  a  spherical  or  pear-shaped  capsule,  the  sporangium.  The 
septum 'which  divides  it  from  the  supporting  hypha  bulges  into  the 
sporangium,  forming  the  columella.  Within  the  capsule  innumerable 


THE   PATHOGENIC  FUNGI 


979 


sporangiospores  develop  which  are  freed  by  its  rupture.  Sexual 
reproduction,  which  is  less  frequent,  consists  in  the  fusion  of  the  tips 
or  lateral  processes  of  two  neighboring  hyphae,  which  form  a  large 
spore  covered  with  a  warty  membrane,  known  as  a  Zygospore.  No 
exospores  are  formed  by  the  mucors  but  chlamydospores  are  numerous. 


FIG.  107. — MUCOR  MUCEDO.  1.  Sporagium,  c.  columella,  m.  sporangium  capsule 
sp.  spores.  2.  Columella,  after  bursting  of  sporangium.  3.  Poorly  developed 
sporangia.  4.  Germinating  spore.  5.  Emptying  of  sporangium. 
Brefeld.) 


(After 


The  common  laboratory  contaminants  are  Mucor  mucedo  a  constant  in- 
habitant of  horse  dung,  and  Mucor  pusillus,  which  can  usually  be  obtained 
by  allowing  a  piece  of  moistened  bread  to  stand  in  a  covered  Petri  dish. 

Mucor  corymbifer  (Lichteimia  corymbifera)  differs  from  the  preceding 
species  in  having  pear-shaped  instead  of  spherical  sporangia  born  in  loose 
clusters  on  hyphae  which  are  not  raised  above  the  surface  of  the  medium. 
This  species  is  pathogenic  for  rabbits,  and  has  been  reported  as  the  cause 
of  inflammations  of  the  auditory  canal  and  of  other  infections  in  man. 


ASCOMYCETES 

In  this  group  are  included  all  fungi  which  form  ascospores.  The 
majority  have  a  mycelium  made  up  of  septate  filaments  and  repro- 
duce by  means  of  conidia  which  are  frequently  born  on  characteristic 


980 


THE  HIGHER   BACTERIA,    MOLDS   AND   FUNGI 


structures.    In  the  lower  types  met  with  as  laboratory  contaminants 
ascospore  formation  is  rarely  observed. 

The  Yeasts. — The  simplest  forms  of  ascomycetes  are  the  exoasci  of 
which  the  yeasts  or  saccharomycetes  are  familiar  examples.  These 
develop  on  laboratory  media  as  moist  masses  of  separate  round  or 
oval  cells  usually  from  10  to  20  microns  in  diameter.  These  send 
out  buds  (blastospores)  which  gradually  assume  the  size  of  the 


FIG.  108. — MUCOR  MUCEDO.  Formation  of  zygospore.  1.  Two  branches  coalesc- 
ing. 2  and  3.  Process  of  conjugation.  4.  Ripe  zygospore.  5.  Germination 
of  zygospore.  6  and  7.  Mucor  erectus.  Azygo  sporulation.  No  two  branches 
meet,  but  form  spores  without  conjugation.  8  and  9.  Mucor  tenuis.  Azygo 
sporulation.  The  spores  grow  out  from  side  branches  without  sexual  union. 
(1-5  after  Brefeld;  6-9  after  Banier,  from  Tavel.) 

parent  cells  from  which  they  quickly  separate.  When  grown  on  a 
dry  surface  some  of  the  cells  divide  into  ascospores  which  remain 
for  a  while  enclosed  in  the  membrane  of  the  parent  cell  OF  ascus. 
Typical  yeasts  are  unicellular  but  in  some  species  the  cells  succes- 
sively produced  by  budding  adhere  to  the  parent  cells  and  form 
mycelial  filaments,  which  consist  of  chains  of  round  or  oval  units. 
In  other  cases  the  individual  cells  of  such  a  chain  elongate  and  form 
a  hypha  with  cylindrical  elements. 


THE   PATHOGENIC   FUNGI  981 

The  yeasts  have  been  studied  most  extensively  in  connection  with  fermen- 
tation and  are  industrially  of  great  importance  in  the  production  of  wine 
and  beer.  Much  of  our  knowledge  of  the  life-processes  of  bacteria  is  based 
on  these  early  investigations  of  the  yeasts.  Although  Schwann,  as  early 
as  1837,  recognized  the  fact  that  many  fermentations  could  not  occur  without 
the  presence  of  yeast,  it  was  not  until  considerably  later  that  the  study 
of  such  fermentations  was  put  upon  a  scientific  basis.  The  typical  fermen- 


A 


FIG.  109. — YEAST  CELLS.     Young  culture  unstained.     (After  Zettnow.) 

tative  action  consists  in  the  transformation  of  sugar  into  ethyl  alcohol  accord- 
ing to  the  following  formula': 

C6H12O8  =  2  C2H5OH  -f  2  CO2. 

The  enzyme  by  which  this  fermentation  is  produced  is  known  as  "zymase," 
and  is,  according  to  Buchner,  in  most  cases,  an  endo-enzyme  which  may  be 
procured  from  the  cell  by  expression  in  a  hydraulic  press.  In  addition  to 
this,  however,  the  various  yeasts  also  produce  other  ferments  by  means 
of  which  they  may  split  higher  carbohydrates,  such  as  saccharose,  maltose, 
and  even  starch,  and  prepare  them  for  action  of  the  zymase.  The  manner 
in  which  this  is  accomplished,  and  the  by-products  which  are  formed  during 
the  process,  vary  among  different  species,  and  it  is  for  this  reason  that 
the  employment  of  pure  cultures  is  of  such  great  importance  in  the  wine 
and  beer  industries  where  differences  in  flavor  and  other  qualities  may  be 
directly  dependent  upon  the  particular  species  of  yeast  employed  for  the 


2  Pasteur,  Etudes  sur  la  biere,  Paris,  1876. 


982 


THE   HIGHER   BACTERIA,    MOLDS   AND    FUNGI 


fermentation.  It  is  due  to  the  work  chiefly  of  Pasteur12  and  of  Hansen3 
that  the  beer  and  wine  industries  have  been  carried  on  along  exact  and 
scientific  lines. 

Many  observers  include  in  the  yeast  family  certain  pathogenic  fungi 
causing  thrush  and  blastomycosis.  These  will  be  described  in  the  section 
on  the  hyphomycetes. 

THE  MOLDS. — The  powdery  molds  which  so  frequently  appear  in 
Petri  dish  cultures  and  which  even  grow  through  cotton  plugs  and 


FIG.  110. — PENICILLIUM  GLAU-     FIG.  111. — ASPERGILLUS    GLAUCUS.     m.    Mycelial 


threads,  s.  Sterigmata.  r.  Ascospore.  p. 
Germinating  cond'lum.  A.  Ascus.  (After  de 
Bary.) 


CUM.  A.  Showing  septate 
mycelia.  B .  End  of  a  hyp  ha 
— branching  into  two  coni- 
diophores,  from  which  are 
given  off  the  sterigmata. 
From  the  ends  of  these  are 
developed  the  round  conidia. 
(After  Zopf .) 

invade  tube  cultures  also  belong  to  ihu  ascomycetes.  The  two 
commonest  genera  have  characteristic  conidiophores  which  make 
their  identification  easy. 


'Hansen,  Prac,  Studies  in  Fermentation,  London,  1896, 


THK   PATHOGENIC   FUNGI  983 

In  the  Penicillium  glaucum  (or  crustaceum)  the  fertile  hyphae  show 
numerous  branches  toward  their  upper  extremities  and  terminate  in  a 
radiating  group  of  flask-shaped  cells  (phialides)  from  the  tips  of  which 
chains  of  conidia  develop — the  structure  somewhat  resembling  a  broom. 
Typical  cultures  have  a  dusty  green  color. 

The  Aspergilli  are  equally  common  and  troublesome  contaminants.  They' 
appear  on  culture  media  as  a  white  feltwork  often  thickly  dotted  with  black 
points  becoming  in  older  cultures  diffusely  black  or  yellow  or  green.  The 
conidia  are  born  on  hyphae  which  terminate  in  a  large  rounded  head  from 
whicli  phialides  project  in  all  directions.  From  the  tips  of  these  extend 
chains  of  conidia,  often  so  densely  packed  together  that  the  supporting 
structure  is  hidden  and  the  whole  appears  as  a  spherical  mass  of  pigmented 
spores.  These  mold  are  active  producers  of  oxalic  and  other  organic  acids. 
The  Sterigmocystis  differs  from  the  aspergillus  in  that  secondary  phialides 
each  bearing  a  chain  of  spores  project  from  each  primary  phialide. 

Pathogenicity. — Many  species  of  these  molds  are  pathogenic  for  labora- 
tory animals  if  the  spores  are  injected  intravenously.  A  number  of  human 
infections  have  also  been  ascribed  to  them.  So-called  pulmonary  asperigillosis 
is  a  condition  clinically  resembling  tuberculosis,  in  which  an  aspergillus, 
usually  the  species  fumigatus,  is  found  abundantly  in  the  sputum.  In  most 
cases  the  fungus  merely  invades  tissue  previously  infected  with  tuberculosis, 
in  other  cases  (notably  in  the  infection  which  has  been  described  in  the 
pigeon  feeders  of  Paris  by  Dieulafoy  and  by  Chantemesse  and  Widal)  the 
disease  is  apparently  primary  and  spontaneous  cure  may  result.  Madura 
foot,  a  disease  which  will  be  referred  to  in  discussing  the  actinomyces  group, 
has  been  in  certain  cases  attributed  to  infection  with  these  molds. 

Pinta  or  Carate,  a  disease  of  tropical  America  characterized  by  super- 
ficial colored  patches  on  the  skin  is  thought  to  be  caused  in  some  cases  by 
aspergilli,  in  others  by  a  species  of  trichophyton.  Aspergilli  have  also  been 
reported  as  infecting  the  eye,  nose,  auditory  canal  and  wounds  in  various 
regions. 

HYPHOMYCETES 

(Fungi  imperfecti) 

Most  of  the  fungi  which  have  definitely  been  shown  to  be  the 
cause  of  human  disease  are  rudimentary  types  in  which  one  can 
detect  no  distinctive  reproductive  processes.  This  makes  impossible 
their  classification  in  the  well  defined  groups  of  fungi.  The  thallus 
is,  as  a  rule,  without  characteristic  structure  and  reproduction  is 
by  means  of  simple  conidia.  Certain  types  do  show  close  analogy 
to  saprophytic  fungi  with  specialized  spores.  For  example,  some 


084  THE   HIGHER   BACTERIA,    MOLDS   AND   FUNGT 

strains  of  blastomycetes  grow  as  a  mass  of  round  cells  developing 
by  means  of  blastospores,  which  indicates  a  close  relationship  to 
the  true  yeasts,  and  although  ascospore  formation  has  never  been 
observed  Brumpt  and  others  group  them  among  the  ascomycetes. 
,  Other  strains  producing  the  same  type  of  disease  are  considered  by 
all  to  be  hyphomycetes.  . 

As  the  botanical  classification  of  these  organisms  is  still  the 
subject  of  controversy  it  has  seemed  clearer  for  the  purposes  of 
this  chapter  to  arrange  them  according  to  the  diseases  which  they 
produce.  For  discussion  of  their  botanical  relationships  as  well 
as  for  an  account  the  numerous  species  which  have  been  described, 
one  is  referred  to  the  works  of  Brumpt,4  Castellanni,5  and  Plant.8 
Only  the  more  important  types  can  be  mentioned  here. 


BLASTOMYCOSIS 

Busse7  in  1894  reported  a  case  of  fatal,  generalized  yeast  infec- 
tion beginning  in  an  abscess  of  the  tibia.  Pus  from  the  lesions 
contained  numerous  giant  cells  in  which  he  observed  round  or  oval, 
double-contoured  bodies  surrounded  by  a  wide  capsule.  They  varied 
in  size  from  that  of  a  red  corpuscle  to  that  of  a  liver  cell.  Many 
showed  small  cells  projecting  as  buds  from  the  larger  parasites. 
The  parasite  grew  readily  on  ordinary  media  in  budding  forms, 
often  surrounded  by  a  capsule  as  in  the  tissues.  It  was  pathogenic 
for  laboratory  animals  especially  for  white  mice.  In  glucose  solu- 
tions it  produced  carbon  dioxide  and  alcohol.  In  1896  Gilchrist8 
described  a  similar  organism  isolated  from  a  patient  with  a  severe 
chronic  cutaneous  disease  which  he  described  as  pseudo  lupus  vul- 
garis.  This  organism  also  was  found  in  giant  cells,  was  capsulated, 
and  showed  budding  forms.  In  culture,  however,  it  produced 
mycelia  and  conidia  and  did  not  ferment  glucose.  Oxalic  acid 
crystals  were  seen  in  the  cultures.  In  the  same  year  Curtis9  in 


4  Brumpt,  Precis  de  Parisitologie,  Masson  et  Cie,  Paris. 

6  Chalmers  and  Castellani,  Handbook  of   Tropical   Medicine. 

6  Plant,  H.  C.,  Handb.  d.  Path.  Microorg.,  Kolle  u.  Wassermann,  vol.  V. 

•'Busse,  Centralbl.  f.  Bakteriol.,  I,  1894,  xvi,   175. 

8  Gilchrist,  Bull.  Johns  Hopkins  Hosp.,   1896,  vii. 

•  Curtis,  Ann.  de  1'Inst.  Pasteur,   1896,  x. 


THE  PATHOGENIC  FUNGI  985 

France  isolated  a  similar  parasite  from  a  myxornatous  tumor  of  the 
leg. 

Numerous  cases  of  infection  with  yeast-like  organisms  have  since 
been  described.  In  a  case  reported  by  Zinsser10  the  lesion  was  an 
abscess  of  the  back  involving  the  spine.  The  parasite  corresponded 
morphologically  to  Busse's.  Animal  inoculation  in  rabbits  and 
guinea-pigs  proved  positive  and  the  organism  seemed  to  show  a 
selective  preference  for  the  lungs  and  spleen.  In  the  lungs  of  the 
animals,  especially,  lesions  were  found  with  surprising  regularity 
even  when  the  inoculation  was  made  intraperitoneally. 

Clinically  there  are  two  distinct  types  of  this  disease,  the  blasto- 
mycetic  dermatitis  of  Gilchrist  and  the  systemic  blastomycosis.  The 
former  is  by  far  the  more  common.  Of  the  latter  a  collection  of 
forty-seven  cases  was  made  in  1916. "  The  portal  of  entry  in  many 
of  these  has  seemed  to  be  the  respiratory  tract,  the  earlier  lesions 
being  in  the  lungs.  In  other  cases  the  general  infection  has  followed 
a  cutaneous  lesion.  The  skin  and  subcutaneous  tissues  show  the 
most  numerous  secondary  lesions  but  the  bones,  liver,  spleen,  kidney 
and  brain  have  each  been  involved  in  several  instances. 


BLASTOMYCES  HOMINIS 

(Sacchnromyces  hominis,  Busse;  Cryptococcus  Gilchristi,  Vuillemin; 
Zymonema  Gilchristi,  de  Beurmann  and  Gougerot;  Mycoderma 
dermatitis,  Brumpt ;  Oidium  Hektoenii,  Ricketts) 

The  parasites  causing  this  disease  probably  represent  a  group 
of  allied  organisms  rather  than  an  individual  species.  As  seen  in 
the  tissues  or  exudates  they  have  shown  a  marked  similarity  in 
all  cases.  In  the  abscesses  of  the  generalized  form  they  are  very 
numerous,  in  the  cutaneous  form  somewhat  more  difficult  to  find 
and  are  best  sought  in  the  small  epidermal  abscesses  which  border 
the  lesion.  In  an  unstained  moist  film  of  pus  spread  out  between 
a  cover-slip  and  slide  they  appear  as  round  or  occasionally  oval 
highly  refractive  bodies  containing  granules  of  various  sizes  and 
often  vacuoles  and  surrounded  by  a  hyalin  capsule.  They  vary 
greatly  in  diameter  usually  from  10  to  20  microns.  Budding  forms 

10  Zinsser,  Proc.  New  York  Path.  Soc.,   1907,  vii. 

11  Wade  and  Bel,  Arch.  Int.  Med.,   1916,  xviii,   103. 


986  THE  HIGHER  BACTERIA,    MOLDS  AND   FUNGI 

and  pairs  united  in  a  figure  eight  can  usually  be  found.  They  stand 
out  more  definitely  if  the  pus  is  cleared  by  mixing  with  a  10  per  cent 
potassium  hydrate  solution.  They  stain  irregularly.  As  a  rule 
with  the  stronger  aniline  dyes  they  are  overstained  so  that  the 
details  are  obscured.  With  hemotoxylin  eosine  the  capsule  usually 
remains  unstained  and  the  body  takes  a  pale  stain  with  deep  blue 
granules  but  in  some  cells  the  capsule  may  stain  deeply.  Thionine, 
polychrome  methylene  blue,  and  Wright's  blood  stain  have  been 
recommended.  In  tissue  sections  stained  with  hemotoxylin  and 
eosin,  or  better  with  thionine,  or  methylene  blue  the  clean  cut, 
circular  parasites  are  easily  recognized,  lying,  as  a  rule,  within 
multinuclear  giant  cells. 

Isolation. — The  isolation  of  the  organism  is  rendered  difficult  by  the 
frequent  presence  in  the  lesions  of  bacteria  which  develop  more  rapidly 
than  the  fungus.  Gilchrist  and  Zinsser  encountered  Gram-positive  cocci, 
others  diphtheroid  bacilli.  No  special  methods  for  facilitating  isolation  have 
been  devised  but  success  will  often  attend  painstaking  and  repeated  plating 
of  the  cultures  in  high  dilution.  The  most  favorable  medium  is  glucose 
agar  and  the  organisms  develop  well  at  room  or  at  incubator  temperature. 

Cultural  Characteristics. — On  agar  or  glucose  agar  the  colonies  appear 
after  two  to  four  days  as  minute  glistening  white  hemispherical  spots  which 
are  not  unlike  colonies  of  staphylococcus  albus.  In  older  cultures  the  appear- 
ance of  different  strains  shows  marked  variations,  some  remaining  smooth 
and  pasty,  others  changing  to  a  tough  wrinkled  membrane  firmly  adherent 
to  the  agar,  and  still  others  becoming  covered  with  white  aerial  hyphae.  All 
become  brown  with  age.  In  agar  stab  cultures  the  organisms  show  their 
preference  for  a  well  oxygenated  environment  by  growing  but  slightly  along 
the  course  of  the  stab  and  by  heaping  up  a  thick  creamy  layer  on  the 
surface  of  the  medium.  Most  strains  fail  to  liquefy  gelatine.  In  broth 
cultures  the  medium  remains  clear,  the  organisms  growing  as  a  stringy 
sediment,  as  a  pellicle,  or  as  tufted  masses  in  the  depth  of  the  medium.  On 
blood  serum,  potato  and  bread  growth  is  easily  obtained.  Some  strains 
ferment  carbohydrates  but  most  do  not. 

Morphology. — In  freshly  isolated  cultures  the  growth  consists 
of  large  round  cells  with  blastospores  like  those  seen  in  the  tissues. 
Capsules  are  often  formed.  Most  strains  sooner  or  later  develop 
coarse,  irregular,  branching  mycelial  filaments.  These  are  divided 
by  septa  and  produce  chains  of  arthrospores  and  terminal  or  lateral 
conidia.  Hamburger1'-  in  a  careful  culture  study  of  four  strains 

12  Hamburger,  Jour.  Inf.  Dis.,  1907,  IV,  201. 


THE  PATHOGENIC   FUNGI  987 

from  systemic  infections  was  impressed  with  the  effect  of  the  tem- 
perature of  incubation  on  the  morphology  of  the  organism.  All 
of  his  strains  grown  in  the  incubator  tended  to  multiply  by  budding, 
while  at  room  temperature  all  were  filamentous,  and  two  produced 
aerial  hyphae. 

Pathogenicity.— All  strains  which  have  been  tested  have  proved 
pathogenic  for  laboratory  animals.  White  mice  seem  the  most 
susceptible. 

Classification. — Cultures  from  the  various  cases  have  shown  little  uni- 
formity. It  is  impossible  to  be  certain  that  they  represent  distinct  species 
as  they  are  somewhat  pleomorphic  and  change  their  morphology  with  varying 
cultural  conditions  and  after  long  preservation  on  artificial  media. 

The  most  serious  attempt  to  systematize  their  varying  char- 
acteristics is  that  of  Ricketts.13  He  considered  the  seventeen  strains 
on  which  he  based  his  report  all  to  be  closely  related  species  of  one 
genus.  He  described  three  main  types  under  which  in  spite  of 
minor  differences  he  grouped  the  various  strains. 

His  classification,  is  not  however,  satisfactory.  Stober14  has  observed 
the  three  types  of  agar  growth  to  occur  successively  in  the  same  strain. 

A  definite  classification  of  these  organisms  is  as  yet  impossible  but  there 

seem  to  be  two  types  which  the  various  strains  more  or  less  closely  resemble. 

I.  The  fermenting  type  described  by  Busse  and  called  Cryptococcus  hominis 

by  Vuillemin,  Brumpt  and  Castellani.     These  organisms  resemble  the 

true  yeasts  in  their  morphology  and  zymogenic  properties. 

II.  The  non-fermenting  type  described  by  Gilchrist  and  called  Cryptococcus 

Gilchristi  by  Vuillemin,  Cryptococcus  dermatitidis  by  Castellani  and 

Mycoderma   dermatitis   by   Brumpt.      These   types   usually   produce 

mycelium  and  reproduce  by  means  of  thallospores  and  conidia.    They 

are  related  to  the  sporothrices  and  trichophyta  described  below.   There 

is  no  correlation  between  the  type   of  organism  and  the   type  of 

lesion  it  produces. 

COOCIDIOIDAL    GRANULOMA 

There  is  a  group  of  cases  resembling  closely  systemic  blastomy- 
cosis,  the  first  observation  of  which  was  that  of  Wernicke  and 
Posadas.15 

"Eicketts,  J.  Med.  Res.,  1901,  vi,  373. 

14  Stober,  Arch.  Int.  Med.,  1914,  xiii,  509. 

15  Wernicke ,  E.,  Centralbl.  f.  Bakteriol.,  I,  1892,  xii,  859. 


988  THE  HIGHER  BACTERIA,    MOLDS  AND  FUNGI 

Their  case  presented  numerous  pea  sized  cutaneous  tumors  and 
clinically  resembled  mycosis  fungoides.  In  the  lesions  they  dis- 
covered intracellular  spherical  hyalin  bodies  which  they  thought  to 
be  protozoan  cysts.  These  cysts  were  surrounded  by  hyalin  capsules 
and  some  of  the  larger  forms  contained  a  great  number  of  the 
daughter  cysts.  Later  Rixford  and  Gilchrist16  reported  a  similar 
case  and  since  then  a  number  of  other  cases  have  been  reported, 
most  of  them  from  the  San  Joaquin  Valley,  California. 

Ophuls  described  three  clinical  types:  (1)  with  primary  cutane- 
ous lesions,  and  later  generalization;  (2)  with  primary  pulmonary 
lesions  and  later  generalization  but  no  skin  lesions;  (3)  with  primary 
pulmonary  lesions  and  secondary  subcutaneous  lesions. 

The  disease  runs  a  more  acute  and  severe  course  than  blastomy- 
cosis  and  of  twenty-four  cases  collected  by  MacNeal  and  Taylor, 
but  two  recovered.  The  cutaneous  lesions  consist  of  large  rather 
painless  granulomatous  abscesses;  there  is  usually  marked  lympha- 
denitis and  the  lungs,  bones,  liver,  kidney,  and  meninges  have  been 
found  to  be  involved.  Histologically,  the  lesions  both  in  man  and 
in  experimentally  infected  animals  very  closely  resemble  those  of 
tuberculosis. 

COCCIDIOIDES  IMMITIS 
(Oidium  coccidioides,  Ophuls ;  Mycoderma  Immite,  Brumpt) 

The  distinction  between  this  parasite  and  that  of  blastomyces 
is  recognized  by  Ophuls,17  Wolbach18  and  MacNeal,19  but  no  typical 
cases  have  been  described  by  Europeans.  The  parasite  seen  in  the 
tissues  resembles  the  blastomyces,  but  does  not  show  buds  and  repro- 
duces by  the  formation  of  endopores.  These  appear  as  a  mass  of 
minute  round  bodies— each  of  which  may  be  capsulated — within  the 
membrane  of  the  parent  cell.  The  parasites  vary  greatly  in  size  and 
some  are  much  larger  than  those  usually  found  in  blastomycosis, 
reaching  50  microns  in  diameter. 

Cultures. — The  colonies  appear  in  surface  plants  in  from  two 
to  seven  days  as  small  slightly  raised  disks  distinctly  penetrating 
the  media.  Older  cultures  become  covered  with  a  dusty  white  layer 

"Bixford  and  Gilchrist,  Johns  Hopkins  Hosp.  Reports,  1896,  I. 

"Ophuls,  W.,  J.  Exper.  M.,  1905,  vi,  443. 

18  Wolbach,  J.  M.  Res.,  1904,  N.  S.  viii,  53. 

"MacNeal,  W.  J.,  and  Taylor,  E.  M.,  J.  M.  Res.,  1914,  xxv,  261. 


THE  PATHOGENIC  FUNGI  989 

of  aerial  hypha.  The  cultures  become  brown  with  age.  In  broth 
they  grow  as  a  fluffy  mass  which  sinks  to  the  bottom.  Sugars  are 
not  fermented.  Gelatin  is  slowly  liquefied  and  milk  slowly  pep- 
tonized. 

Morphology. — In  culture  the  spherical  bodies  immediately  throw 
out  filaments  2  to  8  microns  in  diameter  which  are  branched  and 
septate.  In  older  cultures  they  develop  large  chlamydospores  which 
resemble  the  forms  seen  in  culture  and  also  conidia  which  are  usually 
arranged  in  chains  at  the  end  of  the  hyphae.  In  anaerobic  cultures 
in  Noguchi's  ascitic  agar,  rabbit  kidney  medium,  MacNeal  and 
Taylor  observed  the  formation  of  endospores  like  those  found  in 
the  lesions.  The  organism  is  pathogenic  for  dogs,  rabbits  and 
guinea-pigs. 

Cooke20  has  studied  the  immune  reactions  in  a  human  case  of 
this  disease  and  found  precipitins  which  reacted  up  to  a  1 :160 
dilution  of  the  serum  against  extracts  of  the  cultures.  The  serum 
did  not  react  with  extracts  of  cultures  of  blastomyces.  He  was 
unable  to  obtain  complement  fixation  or  positive  skin  reactions 
with  his  extracts.  t 

THRUSH 

Thrush  is  a  localized  disease  of  the  mouth,  occurring  most  fre- 
quently in  children  suffering  from  malnutrition,  but  also  in  cachectic 
adults.  It  is  characterized  by  the  development  of  creamy  patches 
on  an  area  of  catarrhal  inflammation,  usually  on  the  tongue. 

MONILIA  ALBICANS 

(Oidium  albicans,  Robin;  Saccharomyces  albicans,  Rees;  Endomyces 
albicans,  Vuillemin) 

The  microorganism  which  causes  thrush  was  first  described  by 
Langenbeck  in  1839.  It  is  found  abundantly  in  the  false  membrane 
covering  the  lesion  where  it  appears  as  a  mass  of  simple  or  branched 
mycelial  filaments  made  up  of  irregular  units  of  about  four  by 
twenty  microns.  Oval  cells  of  somewhat  larger  diameter  are  also 
found,  attached  to  the  ends  of  the  filaments,  or  lying  free  and 
throwing  out  buds. 

20  Cooke,  J.  V.,  Arch.  Jjjt,  MecL,  1915,  xv,  479. 


990  THE   HIGHER   BACTERIA,    MOLDS   AND   FUNGI 

The  parasite  grows  readily  on  all  ordinary  media.  On  agar  it  forms 
a  creamy  layer,  in  broth  a  flocculent  deposit.  It  forms  gas  on  certain 
sugars.  Most  strains  do  not  liquefy  gelatin  or  clot  milk.  The  mor- 
phology of  the  organism  has  been  most  thoroughly  studied  by  Roux 
and  Linoissier.  Commonly  it  develops  as  a  mass  of  oval  cells  reproducing 
by  budding  but  often,  especially  in  fluid  media  forms  filaments  like  those 
seen  in  the  lesions,  with  terminal  or  lateral  spores.  Large  globoid  chlamy- 


FIG.  112. — THRUSH.     Oidium  albicans,  unstained.     (After  Zettnow.) 

dospores  at  the  end  of  short  lateral  branches  are  also  found.     These  charac- 
teristics have  led  most  authors  to  call  the  parasite  a  monilia. 

Monilia  is  a  rather  ill-defined  genus  of  hyphomycetes  in  which 
are  included  yeast-like  organisms  which  reproduce  by  blastospores 
but  which  also  form  filaments  of  short  irregular  units  which  are 
easily  detached.  The  filaments  give  rise  to  large  oval  cells  fre- 
quently appearing  as  short  terminal  chains  which  are  called  by  some 
conidia,  by  others,  blastospores.  Castellani  includes  in  this  genus 
only  organisms  which  ferment  sugars  with  the  formation  of  gas. 

Several  authors  have  reported  the  presence  of  ascospores  in  cul- 
tures of  the  thrush  fungus  and  have  classed  it  with  the  saccharom- 
yces.  Vuillemin  found  also  endospores  within  the  mycelial  filaments. 
It  seems  probable  that  the  disease  is  not  always  caused  by  the  same 
organism.  Fischer  and  Brebeck  described  two  varieties,  a  large- 
spored  type  with  endospores  which  liquefied  gelatine,  and  a  small- 
spored  type,  without  endospores  which  did  not  liquefy.  Castellani 
concludes  that  several  species  of  moniliae  and  also  members  of 
other  genera  may  cause  thrush.  On  the  other  hand  Fineman21 
studied  five  strains  from  thrush  and  found  them  identical.  They 
were  however  indistinguishable  from  eight  strains  found  incidentally 


nFineman,  B.  C.,  Jtnir.  Inf.  Pis.,  1921,  XXVIII,  185. 


THE   PATHOGENIC   FUNGI  991 

in  throat  cultures  from  diphtheria  suspects.  All  produced  acid 
and  gas  on  dextrose,  levulose  and  maltose ;  acid  only  on  galactose 
and  saccharose;  and  failed  to  ferment  lactose  and  mannite.22 


SPRUE 

Sprue  is  an  important  disease  of  subtropical  countries  which  is 
characterized  by  progressive  wasting  with  profuse  anemia  and  a 
white  frothy  diarrhea.  There  is  an  inflammation  of  the  entire  intes- 
tinal tract  and  the  lesions  on  the  tongue  are  frequently  characteristic. 

MONILIA  PSILOSIS 

Ashford23  has  made  an  elaborate  study  of  cases  occurring  in 
Porto  Rico.  He  isolated  from  the  tongue  and  stools  of  two  hundred 
patients  an  organism  which  he  calls  monilia  psilosis.  This  is  ap- 
parently identical  with  the  monilia  enterica  of  Castellani. 

Ashford 's  monilia  is  a  large  round  organism  4  to  7  microns  in 
diameter  with  a  granular  and  usually  vacuolated  protoplasm.  On 
Sabouraud's  agar  it  grows  as  a  faintly  greenish  creamy  elevated 
mass  with  mycelium  which  usually  penetrates  the  medium. 

In  gelatin  it  invariably  produces  hyphse  which  spread  laterally  from  the 
stab  and  give  the  growth  an  appearance  which  Ashford  describes  as  that 
of  an  inverted  Christmas  tree.  Monilia  Psilosis  produces  acid  and  gas  on 
dextrose,  levulose,  maltose  and  usually  on  saccharose  but  does  not  ferment 
lactose  nor  mannit.  The  fungus  turns  milk  faintly  alkaline  without  further 
change  and  does  not  liquefy  gelatin. 

On  passage  through  laboratory  animals  (rabbits  and  guinea- 
pigs)  it  produces  a  systemic  mycosis  and  gradually  increases  in 
virulence.  With  these  passage  strains  Ashford  could  produce 
stomatitis  and  diarrhea  by  feeding. 

The  etiological  relationship  of  moniliae  to  sprue  is  not  univer- 
sally accepted.  Castellani  has  isolated  six  different  species  from 
the  stools  typical  cases.  He  believes  that  they  are  not  the  primary 
cause  of  the  disease,  though  they  may  be  responsible  for  the  frothy 
diarrhea. 

-  Eoux  and  Linoissier,  Arch,  de  m&l.  exp.  et  (Tanat.  path.  1890,  IT,  62; 
Compt.  rend,  de  Tacad.  d.  sc.,  1889,  109,  752. 

~*  Ashford,  B.  K.,  Am.  J.  Med.  Sci.,  1917,  cliv,  159. 


992  THE  HIGHER   BACTERIA,    MOLDS  AND   FUNGI 

OTHER   YEAST-LIKE   PARASITES 

Other  diseases  caused  by  members  of  the  yeast  family  have  been 
reported  by  a  number  of  observers.  Tokishige24  found  a  very  minute 
type  in  a  skin  disease,  pseudo glanders,  occurring  among  horses  in 
Japan.  Brumpt  states  that  similar  cases  have  been  found  in  many 
countries  and  that  the  infection  may  attack  man. 

According  to  Mesnil,25  the  Jiistoplasma  capsulatus  of  Darling,26 
which  he  found  in  three  cases  of  splenomegaly  in  Panama,  is  closely 
related  to  the  above  parasite  and  should  be  classed  as  a  cryptococcus. 

The  fact  that  blastomycetes  have  frequently  been  found  in  tumor 
tissue  has  led  several  Italian  observers27  to  assume  an  etiological 
relationship  between  these  microorganisms  and  malignant  growths. 
Absolutely  no  satisfactory  evidence  in  favor  of  such  a  belief  has 
been  advanced,  however,  and  the  yeasts  in  these  conditions  must 
be  regarded  as  purely  fortuitous  findings. 

In  considering  the  possible  origin  of  blastomycetic  infections  in 
animals  and  man,  it  is,  of  course,  important  that  we  should  have 
some  knowledge  as  to  the  pathogenic  properties  of  yeasts  met  with 
and  handled  in  daily  life. 

Rabinowich28  has  investigated  the  pathogenic  properties  of  fifty 
different  species  of  yeast  and  among  them  found  only  seven  varieties 
that  had  any  pathogenicity  for  rabbits,  mice  and  guinea-pigs.  In 
most  of  those  successfully  inoculated  the  disease  produced  in  labora- 
tory animals  had  but  very  little  resemblance  to  blastomycetic  infec- 
tious conditions  observed  in  man. 


SPOROTRICHOSIS 

Sporotrichosis  is  a  chronic  infection  usually  limited  to  the  skin, 
the  subcutaneous  tissues,  and  lymphatics,  occasionally  involving  the 
muscles,  bones  and  joints — and  exceptionally  the  viscera.  The  lesions 
resemble  closely  syphilitic  gummata  and  in  typical  cases  occur  in 

24  Tokishige,    Centralbl.  f .  Bakteriol.,  1896,  i,  19. 

25  Mesnil,  Quoted  by  Brumpt,  loc.  cit. 

M  Darling,  S.  T.,  J.  Exper.  Med.,   1901,  11,  515. 

27  San  Felice,  Centralbl.  f .  Bakteriol.,  I,  1902,  xxxi,  Ztschr.  f .  Hyg.,  1895,  xxi, 
1895,  xviii. 

**  Eabinowich,  Ztschr.  Hyg.,  1895,  xxi. 


THE  PATHOGENIC   FUNGI  993 

a  chain  extending  up  the  arm,  connected  by  thickened  lymphatic 
vessels.    They  slowly  soften  and  ulcerate. 

The  first  cases  described  were  those  of  Schenck29  and  of  Hektoen 
and  Perkins30  in  this  country.  It  is  a  rare  infection  here,  but 
twenty-eight  cases  being  reported  up  to  1912.31  In  France  and 
Switzerland  it  is  relatively  common  and  our  present  knowledge  of 
the  disease  is  based  chiefly  on  the  work  of  de  Beurmann  and 
Gougerot.32  It  may  be  caused  by  several  species  of  hyphomycetes 
belonging  to  the  genus  sporotrichum. 

SPOBOTRICHUM 

It  is  difficult  and  often  impossible  to  demonstrate  the  parasites 
by  direct  examination.  When  found  in  smears  of  the  pus,  or  in 
sections  of  the  lesions,  they  appear  as  oval  or  cigar-shaped  cells 
varying  in  length  from  10  to  2  and  in  breadth  from  3  to  1  microns. 
These  are  frequently  within  large  mononuclear  phagocytes.  The 
parasites  may  be  demonstrated  by  clearing  the  pus  with  40  per  cent 
NaOH  or  by  staining  with  thionine  or  other  basic  stains.  They  are 
Gram-positive. 

Cultural  Characteristics. — As  a  rule  the  organism  can  be  found  only  by 
making  cultures  from  the  pus  or  from  the  bloody  fluid  aspirated  from 
firmer  lesions.  Tubes  of  Sabouraud's  test  medium  or  of  4  per  cent 
glucose  agar  should  be  heavily  inoculated  on  the  surface  and  incubated  at 
room  temperature.  Taylor33  recommends  glycerine-glucose-agar  to  which 
acetic  or  citric  acid  (1-1500)  is  added.  The  colonies  appear  in  four  days 
or  more  as  minute  gray  flecks,  soon  surrounded  by  a  delicate  fringe.  The 
centers  as  they  enlarge  become  raised  and  wrinkled  and  darken  to  a  buff, 
or  chocolate,  or  in  some  species  to  a  black  color.  In  flask  cultures  they 
may  attain  a  diameter  of  10  cm.  and  more.  At  the  periphery  is  a  smooth 
flat  zone  with  delicate  radiating  outgrowths,  which,  if  they  reach  the  side 
of  the  tube,  grow  upward  along-  the  dry  glass.  The  surface  is  usually  hard 
and  glistening  but  in  old  cultures  may  show  hairy  or  powdery  outgrowths 
from  the  surface. 

The  sporothrix  grows  on  media  of  very  simple  composition  but  more 

29  Schenck,  B.  R.,  J.  Hopkins  Hosp.  Bull.  1898,  ix,  286. 

80  Hektoen,  L.,  and  Perkins,  C.  F.,  Jour.  Exp.  Med.,  1900,  V,  77. 

81  Hamburger,  W.  W.,  Jour.  Am.  Med.  Assn.,  1912,  LIX,  1590.     (Bibliography.) 

82  de  Beurmann  and  Gougerot,  Les  Sporotrichoses,  Felix  Alcan.,,  Paris,  1912. 
u  Taylor,  Kenneth,  Jour.  Am.  Med.  Assn.,  1913,  LX,  1142. 


994 


THE   HIGHER   BACTERIA,    MOLDS  AND   FUNGI 


luxuriantly  on  those  containing  sugar.  On  glucose  broth  it  forms  a  thick 
membrane.  It  liquefies  glucose  gelatine  but  does  not  clot  or  digest  milk. 
On  hexoses  it  forms  lactic  acid  but  does  not  ferment  mannite  or  dulcite. 
Species  vary  in  their  action  on  disaccharides. 

Morphology. — This  is  best  studied  in  hang  drop  cultures.  The 
growth  is  made  up  of  interlacing,  branching  septate  hyphae.  These 
are  delicate  (about  2  microns)  and  of  uniform  diameter.  The  spores 


FIG.  113. — SPOROTRICHUM  BEURMANNI  IN  HANG  DROP  (Hopkins). 

are  oval  or  pear-shaped  (about  3  by  4  microns)  and  are  found  on 
any  part  of  the  hyphse  to  which  they  are  usually  attached  by  short 
sterigmata.  In  de  Beurmann's  species  they  are  very  numerous  and 
form  thick  clusters  at  the  tips  of  the  hyphae  or  sheaths  along  their 
course.  Chlamydospores  are  also  found. 

Species. — Sporotrichum  Schencki  is  the  species  found  in  most  American 
cases.  The  cultures  are  white  or  slightly  brownish.  They  ferment  lactose 
but  not  saccharose.  The  spores  are  not  numerous  and  have  no  sterigmata. 

Sporotrichum  Beurrnanni  is  the  commonest  species  in  France.  The  cul- 
tures soon  darken  to  a  chocolate  brown.  They  ferment  saccharose  but  not 


THE  PATHOGENIC  FUNGI  995 

lactose.    Spores  are  very  numerous  and  often  provided  with  short  sterigmata. 
Five  other  pathogenic  varieties  are  listed  by  de  Beurmann  and  there  are 
many  saprophytic  species.     Wolbach34  has  described  a  species  isolated  from 
an  arthritis  of  the  knee  which  he  named  Sp.  Councilmanni. 

Pathogenicity. — Spontaneous  infections  due  to  the  sporotrichum 
Beurmanni  have  been  observed  in  rats,  dogs  and  horses.  Experi- 
mentally cultures  have  been  shown  to  be  virulent  for  rats  and  other 
laboratory  animals. 

The  pathogenic  species  seem  capable  also  of  saprophytic  existence. 
De  Beurmann  recovered  his  species  from  the  normal  throats  of 
patients  with  sporotrichosis  and  of  others  who  had  recovered  from 
the  disease.  Gougerot  found  in  oat  grains,  and  in  other  plants 
sporothrices  which  he  regarded  as  identical  with  the  human  species 
and  which  were  virulent  for  rats. 

Immunity. — Widal  and  Abrami35  found  that  suspensions  of 
sporothrix  spores  were  agglutinated  by  the  sera  of  patients  in 
dilutions  1-200  or  above  and  have  used  this  reaction  in  diagnosis. 
They  also  obtained  fixation  of  complement.  Others  report  that 
patients  give  skin  reactions  with  culture  extracts.  In  human  cases 
there  is  no  evidence  of  the  development  of  an  immunity  but  in 
animals  successful  immunization,  both  active  and  passive,  has  been 
reported. 

DISEASES    OP   THE   RINGWORM   GROUP 

There  is  a  group  of  common  and  relatively  trivial  fungus  infec- 
tions called  dermatomycoses.  In  it  are  included  a  number  of  dis- 
eases clinically  distinct,  all  of  which  are,  however,  characterized  by 
the  fact  that  the  invasion  of  the  parasite  rarely  penetrates  deeper 
than  the  epidermis  and  its  appendages — the  hair  and  nails.  The 
fungi  causing  these  infections  are  known  as  dermatophytes.  They 
are  filamentous  organisms  resembling  in  structure  the  hyphomycetes, 
in  which  group  they  are  usually  placed. 

Brumpt,  Castollani  and  others  prefer  to  group  them  with  the  asco- 
mycetes.  This  decision  is  based  partly  on  their  general  structural  resem- 
blance to  these  higher  fungi  but  chiefly  ou  the  observation  of  Matruchot 

31  irolbach,  Sisson  and  Meier,  Jour.  Mecl.  Res.,  1917,  CXXXVI,  337. 
35  Quoted  by  de  Beurmann  and  Gougerot,  loc.  cit. 


996  THE  HIGHER  BACTERIA,    MOLDS  AND  FUNGI 

and  Dassonville  of  aseospore  formation  in  Eidamella  spinosa,  a  parasite 
isolated  from  a  dermatomycosis  of  the  dog.  In  no  other  member  of  the 
group,  however,  have  ascospores  been  found. 

The  fact  is  that  ringworm-like  diseases  may  be  caused  by  various 
fungi  which  show  little  resemblance  to  each  other  and  that  the 
dermatophytes  as  a  group  are  denned  not  by  any  common  botanical 
characteristics  but  by  the  type  of  lesions  they  produce. 

Our  knowledge  of  the  dermatophytes  dates  from  Schoenlein's 
discovery  of  the  favus  fungus  in  1839.  Two  years  later  Gruby 
discovered  the  fungus  of  ringworm  and  distinguished  between  the 
large  and  small  spored  types.  Since  then  many  workers  have  con- 
tributed to  our  knowledge  of  these  parasites  but  by  far  the  most 
thorough  and  extended  investigation  has  been  made  by  Sabouraud.38 

Cultural  Characteristics. — The  more  important  varieties  of 
dermatophytes  have  been  cultivated  and  show  certain  common  char- 
acteristics. All  of  them  grow  as  leathery  masses  of  closely  inter- 
woven hyphse.  From  a  point  of  inoculation  on  solid  media  they 
spread  out  symmetrically  over  the  surface  at  the  same  time  sending 
down  numerous  short  branches  which  penetrate  the  substrate  and 
bind  the  growth  firmly  to  it.  As  the  membranous  disc  extends 
peripherally  the  central  mycelium  continues  to  grow  increasing  in 
thickness  and  forcing  the  less  adherent  portions  upward.  This 
produces  on  the  surface  a  series  of  humps  and  ridges,  with  cor- 
responding hollows  and  grooves  on  the  under  side,  which  often  form 
striking  geometrical  patterns.  The  surface  may  be  smooth  and 
hard  but  most  species  sooner  or  later  develop  a  duvet — a  covering 
of  aerial  hyphse  which  according  to  their  length  give  the  surface 
a  powdery  or  velvety  or  hairy  appearance.  The  majority  develop 
a  yellow  or  brown  pigment  and  some  are  characterized  by  brilliant 
red  and  violet  colors  which  appear  late  and  are  most  marked  in 
those  portions  of  the  membrane  which  are  raised  above  the  surface 
of  the  medium. 

The  rapidity  of  growth  varies  greatly  in  the  different  species  but  their 
evolution  is  always  a  matter  of  weeks.  Some  varieties  such  as  the  microspora 
attain  a  diameter  of  ten  centimeters  or  more.  Others  do  not  extend  beyond 
one  or  two  centimeters  from  the  center,  but  may  pile  up  a  mass  a  centimeter 
in  thickness. 

86  Sabouraud,  E.,  Les  Teigncs,  Masson  et  Cie.,  Paris,  1910. 


THE  PATHOGENIC  FUNGI  097 

On  broth  they  usually  form  a  thick  membrane  which  spreads  over  the 
surface,  but  if  the  fragment  planted  sinks  it  develops  'only  a  delicate  mesh 
of  filaments  in  the  bottom.  On  potato  the  growth  is  less  vigorous  and 
often  moist.  Gelatin  is  liquefied  by  most  strains.  Glucose  and  mannite  are 
usually  completely  consumed  but  no  gas  is  formed  and  the  medium  is  not 
acidified.  Lactose,  saccharose  and  maltose  are  less  favorable  to  growth. 
Like  many  other  fungi  the  dermatophytes  grow  well  on  the  simpler  synthetic 
media  without  peptone  or  protein,  if  glucose  be  added. 

The  optimum  reaction  is  somewhat  more  acid  than  that  for  bacteria, 
about  PH  7.0;  but  they  develop  readily  throughout  a  range  of  reaction  from 
4.0  to  8  and  above.  Although  obligate  aerobes,  they  will  grow  feebly  with 
scanty  oxygen  supply  as  in  the  depth  of  a  broth  culture.  They  develop  well 
at  room  temperature  and  most  species  also  in  the  incubator. 

Morphology. — Morphologically  as  has  been  said  there  is  little 
that  characterizes  the  ringworm  fungi  as  a  group.  In  the  infected 
hairs  or  skin,  they  appear  as  masses  of  spores  or  as  filaments,  the 
latter  often  consisting  of  chains  of  spore-like  cells.  In  culture  the 
growth  is  made  up  of  branching  hyphae  which  are  always  septate 
but  may  be  coarse  or  delicate,  straight  or  crooked,  cylindrical  or 
irregularly  bulging.  Under  favorable  conditions  most  species  pro- 
duce conidia.  Chlamydospores  are  far  more  common  and  arthro- 
spores  are  also  found.  From  the  common  molds  which  they  some- 
what'resemble  these  parasites  are  distinguished  by  the  absence  of 
ascospores  or  of  the  specialized  conidiophores  such  as  are  found  in 
the  aspergilli  and  penicillii.  These  characteristics  do  not,  how- 
ever, serve  to  distinguish  them  from  other  hyphomycetes. 

Individual  species  do  develop  in  culture  peculiar  spores  and 
mycelial  structures  which  help  to  distinguish  them.  The  following 
are  some  to  which  Sabouraud  has  given  descriptive  names : 

Morphological  Definitions. — Clubs. — These  are  swollen  mycelial  tips  which 
vary  greatly  in  size.  They  are  not  markedly  differentiated  from  the  hyphae 
which  bear  them  but  when  occurring  in  great  numbers  as  in  the  achorion 
of  favus  they  present  a  striking  and  characteristic  appearance. 

Fuseaux. — These  are  large  elongated  chambered  bodies  considered  as 
chlamydospores  by  some,  by  others  as  specialized  conidia.  Sabouraud  applies 
this  name  to  two  different  types,  one  a  lenticular  spore  with  a  tapering 
pointed  tip  and  thick  doubly  contoured  wall,  covered  with  warty  or  hairy 
outgrowths,  the  other  a  blunt  club-shaped  spore  with  thin  smooth  walls.  Both 
are  divided  into  segments  by  parallel  transverse  septa. 

Conidia. — These  are  irregular  in  shape,  size,  and  arrangement.     In  the 


998  THE   HIGHER   BACTERIA,   MOLDS  AND   FUNGI 

microspora  they  are  attached  to  the  fertile  hyphae  by  a  flattened  facet  so 
that  they  resemble  abortive  branches  (Aeladium  type).  In  the  tricophyta 
they  are  more  frequently  attached  by  a  pointed  tip  (Botrytis  type).  Often 
they  are  scattered  irregularly  along  the  hyphae,  but  occasionally  show  a 
characteristic  arrangement.  Groups  of  conidia  formed  along  the  sides  of 
an  unbranched  terminal  hypha  Sabouraud  calls  thyrses,  larger  groups  born 
on  branched  conidiophore  clusters  (grappes}. 

Pectinate  bodies  are  swollen  and  usually  curved  ends  of  hyphae  which 
give  off  a  row  of  abortive  branches  from  one  side — the  structure  vaguely 
resembling  a  comb. 

Spirals. — These  are  simply  convoluted  hyphae  which  may  take  all  the 
forms  of  a  tendril  from  a  spirillum-like  form  to  a  close  set  coil. 

Nodular  organs. — These  are  chains  of  large  rounded  cells  knotted  together 
into  small  dense  masses  suggesting  the  sclerotia  of  higher  fungi. 

Pleomorphism. — The  gross  appearance  and  microscopic  structure 
of  the  dermatophytes  varies  greatly  with  the  cultural  conditions. 
After  a  period  of  growth  on  sugar  containing  media  they  seem 
to  undergo  a  permanent  change,  ceasing  to  produce  conidia  and 
developing  a  thick  covering  of  very  long  aerial  hyphae.  This  gives 
to  these  altered  cultures  a  smooth  white  downy  appearance. 
Sabouraud  refers  to  cultures  of  this  type  as  Pleomorphic  using  the 
term  here  in  a  special  sense.  The  altered  forms  of  different  species 
resemble  each  other  closely  so  that  the  identification  of  a  culture 
by  its  gross  or  microscopic  appearance  is  often  almost  impossible 
once  this  change  has  taken  place. 

Pathogenicity. — The  ringworm  fungi  are  found  with  such 
regularity  and  in  such  profusion  in  many  of  the  human  lesions  as 
to  leave  little  doubt  as  to  their  causative  relationship  to  the  disease. 
Many  of  the  same  species  are  found,  too,  in  similar  lesions  of 
domestic  animals.  These  latter  will  also  induce  lesions  in  guinea- 
pigs  with  more  or  less  regularity  if  culture  fragments  are  inserted 
in  the  skin.  Such  experimental  lesions  are  often  transitory  but 
resemble  somewhat  the  spontaneous  disease.  Other  species  such  as 
the  Microsporon  Audouim  and  Epidermophyton  inguinale,  mentioned 
below,  although  found  abundantly  in  characteristic  human  lesions, 
have  not  been  found  in  animals  and  are  innocuous  when  experi- 
mentally injected. 

Concerning  the  power  of  the  dermatophytes  to  produce  systemic  disease 
we  have  little  information.  Their  entire  lack  of  invasive  power  in  the 
spontaneous  infections  is  quite  striking.  The  isolated  reports  of  Sabrazes 


THE   PATHOGENIC   FUNGI  999 

and  of  Stavino  on  the  production  of  tuberculoid  lesions  by  intravenous 
injections  of  cultures  of  these  fungi  have  little  significance  in  view  of  the 
fact  that  many  types  of  foreign  bodies  produce  similar  results  when  so 
introduced. 

Immunity. — Jadassohn37  has  noted  that  patients  who  have  re- 
covered from  deep  ringworm  infections  seem  immune  from  subse- 
quent attacks.  A  number  of  experimental  observations  especially 
those  of  Bloch  and  Massini88  and  of  Kusunoki39  bear  this  out.  It 
appears  that  animals  which  have  been  infected  with  the  more 
virulent  types  which  produce  suppurative  lesions  resist  reinoculation 
and  that  the  protection  is  valid  against  other  species  than  those 
first  injected.  On  the  other  hand  both  clinical  and  experimental 
observations  show  that  infections  with  less  virulent  species  induce 
no  immunity. 

All  attempts  to  immunize  animals  by  the  injections  of  killed  cultures  and 
extracts  have  failed.  The  reports40  on  the  favorable  therapeutic  action  of 
vaccines  are  inconclusive  on  account  of  the  variable  course  of  the  disease. 
Plato41  was  able  to  produce  in  patients  with  suppurative  ringworm  both 
cutaneous  and  generalized  reactions  by  the  injection  of  extracts  of  trichophyton 
cultures.  Skin  reactions  have  also  been  obtained  in  animals  and  seem  to  be 
nonspecific  in  regard  to  the  species  of  fungus  concerned  but  are,  as  a  rule, 
seen  only  in  animals  with  those  more  severe  infections  which  confer  immunity. 
Kolmer  and  Strickler42  report  that  serum  from  ringworm  cases  gives  comple- 
ment fixation  with  extracts  of  cultures  but  that  the  reactions  were  again 
not  highly  specific  as  to  species. 

Methods  of  Examination. — The  demonstration  of  the  spores  and  mycelium 
in  lesions  is  most  readily  made  by  placing  the  infected  hairs  or  scrapings 
from  the  skin  in  caustic  soda  or  potash  and  covering  with  a  cover  slip.  The 
alkali  renders  the  hair  and  epidermis  transparent  but  does  not  attack  the 
fungus  and  renders  it  easily  visible.  (A  satisfactory  solution  for  this  purpose 
is  a  mixture  of  equal  parts  30  per  cent  aqueous  sodium  hydroxide  and 
glycerin.) 

It  is  possible  to  stain  the  parasites  with  borax  methylene  blue  and  by 
modifications  of  Gram's  method,  but  none  of  the  staining  methods  suggested 

37  Quoted  by  Sabouraud. 

38  Bloch,  B.,  and  Massini,  E.,  Ztschr.  f.  Hyg.,  1909,  Ixxiii,  68. 

39  Kusunoki,  F.,  Arch.  f.  Dermatol.  u.  Syph.,  1912,  cxiv,  1. 
4(1  Strickler,  Jour.  Am.  Med.  Assn.,  Ixv,  225. 

"Keported  by  Neisser,  Arch.  f.  Dermatol.  u.  Syph.,  1902,  lx,  65. 
**  Kolmer  and  Strickler,  Jour.  Am.  Med.  Assn.,  1915, 


1000  THE  HIGHER  BACTERIA,   MOLDS  AND  FUNGI 

have  proved  as  satisfactory  for  diagnosis  as  the  simple  treatment  with 
caustic  soda.  The  process  of  clearing  may  be  hastened  by  gently  heating 
the  slide. 

Cultivation. — Cultures  may  be  obtained  from  the  lesions  by  distributing 
fragments  of  the  infected  epidermis  or  hairs  over  the  surface  of  agar  slants. 
Four  per  cent  glucose  agar  or  Sabouraud's  test  medium  mentioned  below 
are  suitable  for  this  purpose  but  cultures  can  also  be  obtained  on  plain  agar. 
The  chief  difficulty  is  to  avoid  overgrowth  of  the  parasites  by  bacteria  and 
molds.  The  ordinary  plating  procedures  are  inapplicable.  Skin  fragments 
may  be  soaked  for  a  few  minutes,  and  hairs  for  hours,  in  95  per  cent  alcohol 
before  planting  without  killing  the  fungi.  Increasing  the  acidity  of  the 
media  to  PH  5.0 ;  adding  to  it  1  part  in  80,000  of  methyl  violet,  or  1-200,000 
brilliant  green  are  of  some  assistance.  Any  of  these  procedures  inhibit  the 
bacteria  considerably  but  they  are  of  little  avail  against  the  molds.  Main 
reliance  must  be  placed  on  making  a  large  number  of  plants  from  each  case. 

As  the  gross  appearance  of  the  cultures  varies  greatly  with  the  cultural 
conditions,  Sabouraud  has  suggested  a  standard  test  medmm  on  which 
cultures  should  be  planted  for  the  purpose  of  identification.  This  has  the 
following  composition: 

( Sabouraud 's  Test  Medium) 

Maltose  brute  de  Chanut 40  gm. 

Peptone  granulee  de  Chassaing 10  gm. 

Distilled  water    1000  gm. 

Agar  agar    18  gm. 

The  mixture  is  dissolved  in  the  autoclave,  filtered  through  paper,  poured 
into  Erlenmeyer  flasks  to  a  depth  of  about  1  cm.  and  sterilized  once  in 
the  autoclave,  allowing  the  temperature  to  rise  slowly  to  120  degrees. 
Sabouraud  does  not  define  the  reaction  of  the  medium  and  it  cannot  be 
exactly  reproduced  except  by  employing  the  brands  of  reagents  which  he 
suggests.  It  has,  however,  a  reaction  of  about  PH  5.5  and  similar  though 
not  identical  results  can  be  obtained  by  employing  domestic  peptone  and 
malt  extract  and  adjusting  the  reaction  to  this  point. 

In  order  to  prevent  the  pleomorphic  changes  in  the  cultures,  Sabouraud 
recommends  that  stock  strains  should  be  preserved  on  a  medium  of  the 
following  composition: 

(Habourand  's  Conservation  Medium) 

Peptone  granulee   (10  to  50)   usually 30  gm. 

Agar  agar   18  gm. 

Distilled  water 1000  gm. 


THE  PATHOGENIC  FUNGI  1001 

The  study  of  the  morphology  of  cultures  is  made  difficult  by 
the  fact  that  in  detaching  pieces  for  observation  from  the  tough 
mass  of  growth  the  spores  are  usually  detached  and  characteristic 
mycelial  structures  deformed  or  broken  up.  Certain  points  can  be 
made  out  by  examining  such  fragments  mounted  in  water  in  an 
unstained  condition  but  more  information  can  be  gained  from  the 
study  of  hang-drop  cultures.  These  are  made  by  placing  a  large 
drop  of  4  per  cent  maltose  broth  on  a  sterile  cover  slip  inoculating 
it  with  a  fragment  of  the  culture  and  inverting  over  a  hollow  slide 
with  a  deep  concavity.  This  may  be  sealed  with  sterile  oil  or 
petrolatum. 

FAVUS 

Favus  is  a  disease  usually  limited  to  the  scalp  but  which  occasion- 
ally attacks  the  glabrous  skin  and  the  nails.  It  is  characterized  by 
the  formation  at  the  mouths  of  the  hair  follicles  of  small  yellow  cup- 
shaped  crusts  known  as  scutula.  It  is  found  in  adults  as  well  as  in 
children  and  in  this  country  occurs  chiefly  among  immigrants  from 
eastern  and  southern  Europe  where  it  is  endemic. 

ACHORION    SCHOENLEINI 

Favus  of  the  scalp  is  caused  by  one  species  of  fungus  the 
Achorion  Schoenleini.  A  scrutulum  when  crushed  in  alkali  is  found 


FIG.  114. — ACHORION  SCHOENLEINII.     Section  of  favus  crust.     Stained  by  Gram 
(After  Fraenkel  and  Pfeiffer.) 

to  be  composed  almost  entirely  of  the  fungus.  The  central  portion 
is  made  up  of  rounded  sporelike  bodies  of  varying  size  without 
definite  arrangement.  Toward  the  periphery  similar  elements  are 


1002  THE  HIGHER  BACTERIA,   MOLDS  AND  FUNGI 

seen  strung  out  in  filaments,  and  mixed  with  them  hyphae  of  thicker 
elongated  elements  with  irregular  contours.  Within  the  diseased 
hairs  are  filaments,  sometimes  of  cubical,  sometimes  of  elongated 
elements.  They  differ  from  those  found  in  the  hairs  of  ringworm, 
chiefly  in  that  cells  of  different  sizes  and  forms  are  found  in  the 
same  case. 

The  isolation  of  the  achorion  is  rendered  difficult  by  its  frequent  ass'ocia- 
tion  in  lesions  with  pyogenic  cocci  and  molds.  It  develops  slowly  on  agar 
and  the  growth  attains  a  maximum  diameter  of  2  to  3  cm.  in  three  to  four 
weeks.  It  forms  a  remarkably  tough  brownish  membrane  with  deep  irregular 
folds  the  general  outline  being  rounded  upward  toward  the  center.  The 


FIG.  115. — ACHORION  SCHOENLEINI   (Eight  Weeks  Culture  on  Sabouraud's  Test 
Medium  ^  Natural  Size.     Hopkins.) 

surface  is  waxy  at  first  and  later  shows  a  whitish  powdery  duvet.  In  most 
strains  however  after  long  cultivation  on  artificial  media  subcultures  grow 
more  rapidly,  attain  a  larger  size  and  become  covered  with  a  white  velvety 
layer  of  aerial  hyphae  (pleomorphism).  On  gelatin  a  small  surface  growth 
quickly  fluidifies  the  entire  tube  but  the  achorion  utilizes  sugars  slightly  if 
at  all. 

Microscopically  the  growth  is  made  up  of  crooked  hyphae  of 
irregular  contour  often  made  up  of  chains  of  oval  cells.  Pear- 
shaped  conidia  may  be  found  scattered  on  the  sides  of  the  more 
delicate  filaments  but  never  in  clusters.  Chlamydospores  are  always 
numerous  sometimes  attached  singly  to  the  clubbed  tips  of  hyphae 
but  more  often  occurring  in  chains  in  their  course.  These  chains 
may  be  knotted  into  a  small  nodular  mass. 

About  the  periphery  of  a  culture  are  numerous  clubs  either  pear- 
shaped  or  notched  at  the  tip.  These  may  occur  singly  (tetes  de 
clous)  or  in  clusters  (chandeliers  fa viques). 

Inoculation  of  fragments  of  scutula  into  the  skin  of  guinea-pigs 


THE   PATHOGENIC   FUNGI  1003 

produces  transitory  ringworm  like  lesions.  With  some  culture  strains 
similar  results  have  been  obtained  with  others  inoculation  has  been 
unsuccessful. 

Other  Species  of  Achoria. — Fungi  have  also  been  cultivated  from 
favus-like  lesions  in  animals.  The  Achorion  Quinckeanum  which 
frequently  infects  house  mice  has  occasionally  been  found  in  human 
cases  of  favus  of  the  body.  In  culture  these  so-called  achoria  show 
little  in  common  with  Schoenlein's  parasite  but  resemble  somewhat 
trichophyta  of  the  gypseum  group  (V.  infra).  Plant  concludes  that 
there  is  no  reason  for  placing  them  in  this  genus  except  that  they 
form  scutula  in  lesions. 

RINGWORM   OR   TINEA 

The  common  form  of  this  disease  is  tinea  of  the  scalp  which 
affects  only  children.  It  is  highly  contagious  and  in  children's 
schools  and  institutions  may  assume  epidemic  proportions.  The 
infection  begins  in  the  epidermis  where  it  is  often  transitory  but 
the  parasite  soon  invades  the  hairs  and  there  remains. 

There  is  usually  slight  evidence  of  inflammation  but  some  species 
of  the  ringworm  fungi  cause  suppuration  in  and  about  the  hair 
follicle.  This  may  result  in  the  formation  of  large  indolent  subcu- 
taneous abscesses  known  as  kerions. 

Tinea  of  the  body  may  occur  secondarily  to  scalp  lesions  in 
children  or  as  a  primary  infection  at  any  period  of  life.  The  lesions 
often  assume  a  circular  form — the  disease  progressing  at  the  per- 
iphery and  clearing  at  the  center.  There  may  be  only  slight  thicken- 
ing and  desquamation  of  the  epidermis  but  usually  there  are  super- 
ficial vesicles  which  quickly  dry  into  crusts,  and  occasionally 
follicular  pustules  and  kerions.  Some  species  invade  the  nails. 

Ringworm  is  also  a  common  disease  in  domestic  animals  and 
human  cases  can  often  be  traced  to  infection  from  these  sources. 
The  fungi  of  animal  origin  when  they  infect  man  either  directly 
from  a  diseased  animal  or  indirectly  from  another  human  case, 
usually  produce  more  inflammatory  lesions  than  do  those  species 
which  affect  man  only. 

MICROSPORON 

The  small  sporod  ringworm  fungi  are  in  this  locality  the  com- 
monest cause  of  ringworm  of  the  scalp.  The  species  of  animal 


1004  THE  HIGHER  BACTERIA,    MOLDS  AND  FUNGI 

origin  also  attack  the  glabrous  skin  especially  in  children.  In  the 
epidermis  of  the  diseased  scalp  they  appear  as  curved  branching 
hyphae  made  up  of  elongated  elements.  The  stumps  of  the  diseased 
hairs  are  covered  with  a  mosaic  of  small  round  spores  of  uniform 
diameter  (about  2  microns)  which  completely  envelops  the  hair. 
If  the  hair  is  crushed,  mycelial  threads  are  also  seen  which  grow 
along  the  medulla.  From  these  central  filaments  branches  project 
out  through  the  cortex  and  give  rise  to  the  sheath  of  spores. 

Cultures. — These  parasites  are  easily  cultivated.  They  grow 
rapidly  on  agar  producing  large  flat  colonies  which  from  the  first 
are  covered  with  a  duvet  of  aerial  hyphaB.  At  the  center  is  a  raised 
papilla  and  from  this  folds  in  the  membrane  radiate  out.  The  color 
of  the  duvet  varies  from  snow-white  to  deep  buff  and  of  the  mem- 


Fio.  116. — MICROSPORON  LANOSUM  (Six  Weeks  Culture  on  Sabouraud's  Test  Medium 
\  Natural  Size.     Hopkins). 

brane  from  buff  to  brilliant  orange  or  even  a  russet  brown.  The 
pigment,  which  is  well  developed  only  on  glucose-containing  media, 
is  diffusable.  Gelatin  is  very  slowly  liquefied. 

Morphology. — In  young  cultures,  long  straight  coarse  trunks 
radiate  out  from  the  center  giving  off  frequent  branches  which 
later  form  an  inextricable  tangle  of  threads  running  in  all  directions. 
Some  terminal  branches  bear  conidia  attached  to  their  sides  by  a 
flattened  facets  (Acladium  type).  They  also  form  chlamydospores, 
and  fuseaux. 

Sabouraud  describes  eleven  species  of  microspora  which  he  divides  into 
two  groups :  one  affecting  man  only  and  one  affecting  both  animals  and  man. 

Microsporon  Audouini. — This  is  the  type  species  of  the  former  group. 
In  gross  appearance  the  cultures  are  distinguished  by  their  slower  growth, 


THE  PATHOGENIC  FUNGI 


1005 


their  velvety  white  or  faintly  buff  duvet  and  less  active  pigment  production. 
Microsporon  Audouini  produces  only  occasional  and  atypical  fuseaux  but 
numerous  chalmydospores  which  are  frequently  sub-terminal  the  hypha  pro- 
jecting beyond  them  like  a  spine.  It  also  produces  typical  pectinate  bodies. 
Animal  inoculation  has  been  unsuccessful.  Closely  allied  to  this  species  are: 
Microsporon  umbonatum,  M.  tardum,  and  M.  velveticum. 

Microsporon  lanosum  (M.  Audouini,  var.  cams). — This  is  the  type  species 
of  the  microspora  of  animal  origin.  It  produces  ringworm  in  the  dog.  The 
cultures  grow  very  rapidly  and  on  glucose  are  deeply  pigmented.  The  duvet 
is  long,  shaggy,  and  in  older  growths  assumes  a  dark  tan  color.  The  upper 


FIG.  117. — FUSEAUX  OF  MICROSPORON  LANOSUM X200. 

surface  is  covered  with  a  thick  crop  of  typical  lenticular  fuseaux  which  alone 
serve  to  identify  cultures  as  belonging  to  this  group.  Experimental  lesions 
in  the  guinea-pig  can  be  produced  by  the  inoculation  of  cultures.  Other 
members  of  this  group  are:  Microsporon  felineum,  M.  equinum,  M.  fulvum, 
M.  villosum,  M.  pubescens,  and  M.  tomentosum. 

TRICHOPHYTON 

The  trichophyta,  like  the  microspora,  may  produce  ringworm  of 
the  scalp  but  they  also  produce  lesions  on  the  glabrous  skin  and 
nails.  The  genus  is  defined  chiefly  by  the  fact  that  its  members 
produce  lesions  without  scutula  and  appear  in  infected  hairs  as 
chains  of  spore-like  elements.  This  alignment  of  the  so-called  spores 
(which  are  in  reality  short  mycclial  elements)  distinguishes  them 
from  those  of  the  preceding  genus.  In  most  trichophyta,  too,  the 
spores  are  large  but  in  a  few  they  are  of  about  the  same  size  as 
those  of  the  microspora  (3  microns).  In  culture  most  species  produce 
conidia  attached  to  the  hyphae  by  pointed  tips  and  frequently 


1006  THE  HIGHER   BACTERIA,    MOLDS   AND   FUNGI 

arranged  in  clusters.  A  few  species  (Tr.  violaceum)  which  bear  no 
conidia  are,  however,  included  in  the  genus.  On  artificial  media 
the  growths  are  much  buckled  and  raised  as  compared  to  the  flat 
cultures  of  the  microspora.  The  cultures,  however,  vary  so  greatly 
among  themselves  in  appearance  that  no  description  can  be  given 
which  would  apply  to  the  genus  as  a  whole. 

Classification. — Sabouraud  lists  thirty  species.  His  classification  is  based 
primarily  on  the  appearance  of  the  parasite  in  the  infected  hairs.  Such 
a  scheme  has  the  disadvantage  that  it  groups  together  forms  which  have 
diverse  cultural  characteristics  and  separates  others  which  culturally  are 
similar.  He  divides  them  first  into  three  divisions : 

I.  The  Trichophyton  endothrix  in  which  the  fungus  is  found  only  within 

the  medulla  of  the  hair. 

II.  The  Trichophyton  neo-endothrix  in  which  some  infected  hairs  show 

also  a  few  filaments  on  their  surface. 

III.  The  Trichophyton  ectothrix  in  which  besides  invading  the  substance 

of  the  hair  the  fungus  proliferates  actively  on  its  surface. 
Of  this  third  division  there  are  two  groups : 

A.  The  microid  endothrices,  of  which  the  spore-like  elements  about  the 
infected  hairs  are  from  3  to  4  microns  in  diameter.     They  are  again  divided 
into  two  sub-groups,  differing  culturally:   (a)   The  Gypseum  Group  and  (b) 
The  Niveum  Group. 

B.  The  megalospora,   of  which  the  rounded   elements  are   from   6  to  8 
microns  in  diameter.     These   Sabouraud  again   divides  into:    (a)    A  group 
forming  downy  cultures,  and  (b)  A  group  in  which  the  cultures  are  faviform. 

The  typical  members  of  the  Endothrix  Group  (Tr.  crateriforme  and 
Tr.  acuminatum)  form  small  raised  colonies  less  than  4  cm.  in  diameter. 
They  are  white  or  slightly  yellowish  and  are  covered  from  the  first  with 
a  short  powdery  or  velvety  duvet  but  show  little  tendency  to  pleomorphic 
change.  They  produce  numerous  conidia  in  thyrses  or  small  clusters  but 
show  no  other  characteristic  structures.  Sabouraud  describes  many  variants 
of  these  species  which  differ  in  the  contour  of  their  colonies  or  in  pigment 
production.  They  are  frequently  found  in  mild  ringworm  of  the  scalp  in 
France  but  so  far  we  have  not  found  them  in  New  York. 

Trichophyton  violaceum,  which  is  also  an  endothrix,  is  a  very  different 
organism.  It  develops  slowly,  forming  small  wrinkled  colonies  with  a  hard 
glistening  surface,  which  slowly  develop  a  black-violet  color.  The  mycelium 
is  made  up  of  short  crooked  elements  and  no  conidia  are  formed.  It  is 
found  with  relative  frequency  in  tinea  of  the  scalp,  body  and  nails.  A 
variant  (Tr.  glabrum)  differs  only  in  the  absence  of  pigment. 

Trichophyton  cerebriforme,  the  type  of  the  Neo-Endothrix,  Group,  re- 
sembles in  culture  the  Tr.  crateriforme. 


THE   PATHOGENIC   FUNGI  1007 

The  Gypseuni  Group  (Tr.  asteroides,  radiolatum,  etc.)  grow  more  vigor- 
ously forming  large  colonies  up  to  10  cm.  in  diameter.  The  surface  is 
powdery  or  plaster-like,  but  the  parasites  soon  become  pleomorphic  and 
produce  a  long  velvety  duvet.  They  produce  conidia  in  large  clusters,  rudi- 
mentary fuseaux,  and  numerous  spirals.  The  lesions  are  inflammatory,  with 
folliculitis  and  kerion  formation.  They  cause  ring-worm  in  horses. 

The  Niveurn  Group  resemble  the  pleomorphic  forms  of  the  gypseurn 
group.  They  form  conidia  only. 

Trichophyton  rosaceum  (the  type  of  the  Downy  Megalospora)  forms  a 
colony  of  medium  size  resembling  a  folded  disc  of  white  velvet.  The  deep 
portion  develops  a  crimson  or  violet  pigment  which  gives  a  rose  tint  as 
seen  through  the  white  duvet.  It  forms  long  thyrses  and  rudimentary  fuseaux. 

The  Faviform  Strains  (Tr.  ochraceum,  Tr.  album,  etc.)  resemble  culturally 
the  Achorion  Schoenleini.  They  are  grouped  with  the  trichophyta  chiefly 
because  they  form  no  scutula  in  lesions. 

A  description  of  all  the  individual  species  would  exceed  the  scope 
of  this  chapter. 

ECZEMA    MARGINATUM   AND    POMPHOLYX 

Eczema  marginatum  or  ringworm  of  the  groin  is  a  common  derma- 
tosis.  In  the  lesions  Castellan!  and  Sabouraud  found  a  fungus  to 
which  the  latter  gave  the  name  Epidermophyton  inguinale.  More 
recently  Ormsby  and  Mitchell43  and  others  in  this  country  have 
found  the  same  fungus  in  eczematous  lesions  of  the  hands  and  feet 
and  also  in  the  vesicular  eruptions  in  these  regions  formerly  called 
pompholyx.  Sabouraud  has  also  found  various  species  of  trichophyta 
in  palmar  eczemas. 

EPIDERMOPHYTON  INGUINALE 

(Trichophyton  cruris) 

In  the  epidermis  the  parasite  is  seen  as  long  interlacing  filaments 
made  up  of  oblong  or  oval  cells  with  double  contours.  It  develops 
slowly  in  culture  as  a  greenish  buff  colony  with  folds  radiating  from 
a  central  or  slightly  eccentric  peak,  attaining  a  diameter  of  perhaps 
two  centimeters  in  a  month.  The  surface  at  first  is  dry  and  powdery 
but  on  sugar  media  it  quickly  becomes  pleomorphic,  developing  a 

43  Ormsby,  0.  8.,  and  Mitchell,  J.  H.,  Jour.  Am.  Med.  Assn.,  1916,  LXVII,  711. 


1008 


THE   HIGHER   BACTERIA,    MOLDS  AND  FUNGI 


thick  white  duvet.     Related  varieties  are  also  found  which  form 
huge  crateriform  colonies. 

The  cultures  form  no  conidia  but  are  readily  identified  by  the 
innumerable  blunt  fuseaux  which  are  born  on  aerial  hyphge.  These 
are  often  in  clusters  which  have  been  compared  to  a  hand  of 
bananas.  Older  cultures  show  also  many  intercalary  chlamydospores. 
The  fuseaux  are  not  found  in  cultures  which  have  become  downy. 


t.>&% 


V?- 


FIG.  118. — FUSEAUX  OF  EPIDERMOPHYTON  INGUINALEX200. 

Inoculations   of   epidermophyton   cultures   into   men,    dogs   and 
guinea-pigs  have  been  uniformly  unsuccessful. 


TINEA  VERSICOLOR 

(Pityriasis  versicolor,  Chromophytosis) 

Tinea  versicolor  appears  as  large  fawn-colored  patches  on  the 
covered  parts  of  the  body.  These  show  slight  superficial  scaling 
but  no  evidence  of  inflammation.  The  causative  fungus  is  found 
abundantly  in  the  horny  epidermis  where  it  seems  to  grow  as  a 
saprophyte. 


THE   PATHOGENIC  FUNGI   %  1009 

Microsporon  furfur  (Malassezia  furfur).  This  fungus  bears  little 
resemblance  to  the  microspora  which  cause  ring-worm.  It  appears 
as  septate  filaments  of  very  irregular  contour  3  to  4  microns  wide. 
They  are  usually  unbranched  but  interlace,  forming  a  meshwork 
in  which  are  found  masses  of  spore-like  bodies.  Most  attempts  to 
isolate  the  organism  have  failed.  A  few  investigators44  have 
reported  successful  cultures  but  their  work  lacks  confirmation. 


ERYTHRASMA 

Erythrasma  is  a  superficial  infection  of  the  epidermis  which 
produces  round  scaling  patches  usually  located  in  the  axillae  or 
groins.  There  is  no  evidence  of  inflammatory  reaction  except  a 
hyperemia  which  gives  the  lesions  a  characteristic  red  color.  A 
parasite  is  found  in  the  epidermis  which  appears  as  minute  twisted 
threads  which  are  easily  broken  up  into  elements  about  the  size 
of  bacilli.  Cultures  have  been  described  by  Michele  and  by  Ducrey 
and  Reale  but  according  to  others  the  organism  cannot  be  cultivated. 
It  has  been  given  various  names:  Microsporon  minutissimum,  sporo- 
tJirix  minutissimum,  and  nocardia  minutissimum. 

"Kotjar,  Kef.  Baugartens  Jahresbericht,   1892. 


SECTION  VI 


BACTERIA  IN  AIR,  SOIL,  WATER,  AND  MILK 
CHAPTER   LI 

BACTERIA  IN  THE  AIR  AND  SOIL 

BACTERIA   IN    THE    AIR 

BACTERIA  reach  the  air  largely  from  the  earth's  surface,  borne 
aloft  by  currents  of  air  sweeping  over  dry  places.  Their  presence 
in  air,  therefore,  is  largely  dependent  upon  atmospheric  conditions; 
humidity  and  a  lack  of  wind  decreasing  their  numbers,  dryness  and 
high  winds  increasing  them.  Multiplication  of  bacteria  during 
transit  through  the  air  probably  does  not  take  place. 

Apart  from  these  considerations  the  presence  of  bacteria  in  air 
also  depends  upon  purely  local  conditions  prevailing  in  different 
places.  They  are  most  plentiful  in  densely  populated  areas  and 
within  buildings,  such  as  theaters,  meeting  halls,  and  other  places 
where  large  numbers  of  people  congregate.  On  mountain  tops,  in 
deserts,  over  oceans,  and  in  other  uninhabited  regions,  the  air  is 
comparatively  free  from  bacteria.  A  classical  illustration  of  this 
fact  is  found  in  the  experiments  which  Pasteur  carried  out  in  his 
refutation  of  the  doctrine  of  spontaneous  generation.  Tyndall  also, 
in  working  upon  the  same  subject,  demonstrated  this  fact.  From 
the  surface  of  the  ground  and  other  places  where  bacteria  have  been 
deposited,  they  reach  the  air  only  after  complete  drying.  It  is  a 
fact  of  much  importance,  both  in  bacteriological  work  and  in  sur- 
gery, that  bacteria  do  not  rise  from  a  moist  surface.  From  dry 
surfaces  they  may  rise,  but  only  when  the  air  is  agitated  either 
by  wind  or  by  air-currents  produced  in  other  ways.  In  closed  rooms, 
therefore,  even  when  bacteria  are  plentiful  and  the  walls  and  floors 
are  perfectly  dry,  there  is  little  danger  of  the  inhalation  of  bacteria 
unless  the  air  is  agitated  in  some  way.  The  most  favorable  condi- 

1010 


BACTERIA  IN   THE  AIR  AND  SOIL  1011 

tions  for  the  occurrence  of  many  bacteria  in  air  are  the  existence 
of  a  prolonged  drought  followed  by  a  dry  wind.  Under  such  condi- 
tions, even  the  dark  places  and  unlighted  corners  of  streets  and 
habitations  are  thoroughly  dried  out,  and  bacteria  are  taken  up 
and  carried  about  together  with  particles  of  dust.  At  such  times 
the  dangers  from  inhalation  are  much  multiplied.  By  experiments 
made  in  balloons,  it  has  been  found  that  bacteria  are  plentiful  below 
altitudes  of  about  fifteen  hundred  feet  and  may  be  present,  though 
much  reduced  in  numbers,  as  high  up  as  a  mile  above  the  earth's 
surface.  The  species  of  bacteria  found  in  the  air  are,  of  course, 
subject  to  great  variation,  depending  upon  locality.  Molds  and 
spore-forming  bacteria,  being  more  regularly  resistant  to  the  effects 
of  sunlight  and  drying  than  bacteria  possessing  only  vegetative 
forms,  are  naturally  more  generally  distributed. 

Out  of  air  thus  laden  with  bacteria,  they  may  again  settle  when 
the  wind  subsides  and  the  air  becomes  quiescent.  The  process  of 
settling,  however,  is  extremely  slow,  since  the  weight  of  a  bacterium 
is  probably  less  than  a  billionth  of  a  gram,  and  it  may  be  held  in 
suspension  in  air  for  considerable  periods.  Rains,  snow,  or  even 
the  condensation  of  moisture  from  a  humid  atmosphere,  hastens 
this  process  considerably  and  large  quantities  of  bacteria  may  settle 
out  from  air,  in  a  comparatively  short  time,  in  ice  chests,  in  operating 
rooms,  or  in  other  places  in  which  much  condensation  of  water  vapor 
takes  place. 

The  importance  of  the  air  as  a  means  of  conveying  disease  is 
still  a  problem  upon  which  much  elucidation  is  needed.  The  im- 
portance of  fhis  manner  of  conveyance  in  smallpox,  in  measles,  in 
scarlet  fever,  and  in  other  exanthemata,  can  not  be  denied.  As 
regards  the  diseases  of  known  bacterial  origin,  conveyance  by  air 
is  of  importance  in  the  case  of  tuberculosis,  where  infection  by 
inhalation  may  take  place,  and  in  the  case  of  anthrax,  where  inhaled 
anthrax  spores  may  give  rise  to  the  pulmonary  form  of  the  disease. 
The  importance  of  air  conveyance  for  any  great  distance  in  pneu- 
monia, in  influenza,  in  diphtheria,  and  in  meningitis  is  by  no  means 
clear  and  requires  much  further  study.  The  expulsion  of  bacteria 
from  the  lungs  and  naso-pharynx  does  not  take  place  during  simple 
expiration,  since  an  air-current  passing  over  a  moist  surface  is  not 
sufficient  to  dislodge  microorganisms.  Expulsion  of  bacteria  in 
these  conditions  must  take  place  together  with  small  particles  of 
moisture  carried  out  in  sneezing,  coughing,  or  any  forced  expiration. 


1012  BACTERIA   IN  AIR,   SOIL,   WATER,   AND   MILK 

The  bacteria  thus  discharged  arc  then  subject  to  the  process  of 
drying  and  often  are  exposed  to  direct  sunlight  for  a  considerable 
period  before  they  are  again  taken  up  in  the  air. 

The  methods  of  estimating  the  bacterial  contents  of  the  air  are 
not  entirely  satisfactory.  The  simple  exposure  of  uncovered  gelatin 
or  agar  plates  for  a  definite  length  of  time,  and  subsequent  estimation 
of  the  colonies  upon  the  plates,  yield  a  result  which  is  variable 
according  to  the  air-currents  and  the  degree  of  moisture  in  the 
atmosphere,  and  furnish  no  volume  standard  for  comparative  results. 
The  methods  which  are  in  use  at  the  present  time  depend  upon 
the  suction  of  a  definite  quantity  of  air  by  means  of  a  vacuum-pump 
through  some  substance  which  will  catch  the  bacteria.  One  of  the 
first  devices  used  for  this  purpose  was  that  of  Hesse,  who  sucked 
air  through  a  piece  of  glass  tubing,  about  78  cm.  long  and  about 
3.5  cm.  in  diameter,  the  inner  surface  of  which  had  been  coated 
with  gelatin  in  the  manner  of  an  Esmarch  roll  tube.  This  method 
is  not  efficient,  since  a  large  number  of  the  bacteria  may  pass  entirely 
through  the  tube  without  settling  upon  the  gelatin.  One  of  the 
most  satisfactory  methods  at  present  in  use  is  that  in  which  definite 
volumes  of  air  are  sucked  through  a  sand-filter.  Within  a  small 
glass  tube,  a  layer  of  sterilized  quartz  sand,  about  4  cm.  in  depth, 
is  placed.  The  sand  is  kept  from  being  dislodged  by  a  small  wire 
screen.  After  the  air  has  been,  sucked  through  the  filter  the  sand 
is  washed  in  a  definite  volume  of  sterile  water  or  salt  solution,  and 
measured  fractions  of  this  are  planted  in  agar  or  gelatin  in  Petri 
plates.  The  colonies  which  develop  are  counted.  Thus,  if  two  liters 
of  air  have  been  sucked  through  the  filter,  and  the  sand  has  been 
washed  in  10  c.c.  of  salt  solution,  and  1  c.c.  of  this  is  planted,  with 
the  result  of  fifteen  colonies,  then  the  two  liters  of  air  have  contained 
one  hundred  and  fifty  bacteria. 


BACTERIA    IN   SOIL 

Besides  the  normal  bacterial  inhabitants  of  the  soil,  bacteria 
reach  the  soil  from  the  air,  in  contaminated  waters,  in  the  dejecta, 
excreta,  and  dead  bodies  of  animals  and  human  beings,  and  in  the 
substance  of  decaying  plants.  It  is  self-evident,  therefore,  that  the 
distribution  of  bacteria  in  soil  depends  largely  upon  the  density 
of  population  and  the  use  of  the  soil  for  agricultural  or  other 


BACTERIA  IN   THE  AIR  AND  SOIL  1013 

purposes.  Thus,  bacteria  are  most  plentiful  in  the  neighborhood  of 
cess-pools  or  in  manured  fields  and  gardens.  Such  conditions,  how- 
ever, may  be  regarded  as  abnormal.  Even  in  uncultivated  fields 
there  is  a  constant  bacterial  flora  in  the  soil  which  is  of  great 
importance  in  its  participation  in  the  nitrogen  cycle,  a  phase  of  the 
bacteriology  of  soil  which  has  been  discussed  in  detail  in  another 
section. 

There  are,  thus,  regular  and  normal  inhabitants  of  the  soil  which 
fulfill  a  definite  function  and  may  be  found  wherever  plant  life 
flourishes.  In  addition  to  these,  innumerable  varieties  of  sapro- 
phytes and  pathogenic  germs  may  be  present,  which  vary  in  species 
and  in  number  with  local  conditions.  Numerous  investigations  into 
the  actual  numerical  contents  of  the  soil  have  been  made.  Houston1 
found  an  average  of  1,500,000  bacteria  per  gram  in  garden  soil, 
and  about  100,000  bacteria  per  gram  in  the  arid  soil  of  uncultivated 
regions.  Fraenkel,2  in  studying  the  horizontal  distribution  of  bac- 
teria in  the 'earth,  has  found  that  they  are  most  numerous  near  the 
surface,  a  gradual  diminution  occurring  down  to  a  depth  of  about 
two  yards.  Beyond  this,  the  soil  may  be  often  practically  sterile. 

Pathogenic  bacteria  may  at  times  be  found  in  the  surface  layers, 
and  these  are  often  of  the  spore-bearing  varieties.  Most  important 
among  them  from  the  medical  standpoint  are  the  bacillus  of  tetanus, 
of  malignant  edema,  and  the  Welch  bacillus.  If  a  guinea-pig  is 
inoculated  subcutaneously  with  an  emulsion  of  garden  soil,  death 
will  result  almost  invariably  with  enormous  bloating  and  swelling 
of  the  body  due  to  gas  production.  This  is  due  to  the  fact  that 
the  spore-bearing,  gas-producing  anaerobic  bacilli  are  commonly 
present  and  are  actively  pathogenic  for  these  animals.  The  frequent 
occurrence  of  tetanus  in  persons  sustaining  wounds  of  the  bare  feet 
and  hands  in  fields  and  excavations,  is  a  matter  of  common  knowl- 
edge. Anthrax,  also,  may  be  easily  conveyed  by  soil  in  localities 
where  animals  are  suffering  from  this  infection.  It  is  not  probable 
that  pathogenic  germs  which  are  not  spore-bearers  survive  in  the 
soil  for  any  great  length  of  time.  Unless  the  soil  is  specially  pre- 
pared by  the  presence  of  defecations  or  other  organic  material,  the 
nutrition  at  their  disposal  is  not  at  all  suitable  for  their  needs,  since 
rapid  decomposition  of  organic  materials  by  saprophytes  is  always 


1  Houston,  Report  Mod.  Officer,  Local  Govern.  Bd..  London,  1897 

2  Fraenkel,  Zeit.  f .  Hyg.,  ii,  1887. 


1014  BACTERIA   IN   AIR,   SOIL,    WATER,   AMD    MILK 

going  on  in  the  upper  layers.  Furthermore,  in  the  deeper  layers 
the  conditions  of  temperature  and  possibly  oxygen  supply  are  not 
at  all  favorable  for  the  growth  of  most  pathogenic  bacteria.  Within 
a  short  distance  from  the  surface  the  temperature  of  the  soil  usually 
sinks  below  14°  or  15°  C.'  An  interesting  series  of  experiments  by 
Fraenkel3  have  demonstrated  this  point.  This  investigator  buried 
freshly  inoculated  agar  and  gelatin  cultures  of  cholera  spirilla  and 
of  typhoid  and  anthrax  bacilli  at  different  levels,  and  examined 
them  for  growth  after  two  weeks  had  elapsed.  The  anthrax  bacilli 
hardly  ever  showed  growth  at  a  depth  below  about  two  yards,  and 
cholera  and  typhoid  developed  colonies  at  these  depths  only  during 
the  summer  months.  Under  natural  conditions  it  must  be  remem- 
bered that,  at  these  levels,  suitable  nutritive  material  is  not  found. 
A  consideration  of  practical  importance  in  this  connection  is  the 
possibility  of  infection  by  means  of  buried  cadavers.  An  elaborate 
series  of  experiments  has  been  carried  out  upon  this  subject  in 
Germany,  with  results  which  demonstrate  that  the  danger  from  the 
burial  of  persons  dead  of  infectious  diseases  was  formerly  much 
exaggerated.  Experiments4  usually  failed  to  reveal  the  presence  of 
cholera  and  typhoid  bacilli  within  two  to  three  weeks  after  burial, 
and  tubercle  bacilli  were  never  found  after  three  months  had  elapsed. 
It  was  only  in  the  case  of  sporulating  microorganisms,  such  as  the 
anthrax  bacillus,  that  the  living  incitants  could  be  found  for  as 
long  as  two  years  after  burial.  The  dangers  of  infection  of  human 
being  through  the  agency  of  soil,  therefore,  are  chiefly  those  arising 
from  the  spore-bearing  bacteria  which  are  able  to  remain  alive  in 
spite  of.  the  unfavorable  cultural  conditions.  It  has  been  found 
by  some  observers,5  however,  that,  under  special  conditions,  non- 
sporulating  bacteria,  more  especially  the  typhoid  bacillus,  may 
remain  alive  in  soil  for  several  months.  Although  these  bacteria, 
as  well  as  those  of  cholera,  diphtheria,  etc.,  can  not  proliferate  under 
the  conditions  found  in  the  soil,  the  fact  that  they  can  remain 
viable  for  such  prolonged  periods  in  the  upper  layers  suggests  the 
possibility  of  danger  from  the  use  of  unwashed  vegetables,  such  as 
lettuce  or  radishes  or  other  soil  and  sewage  contaminated  food 
products.  The  examination  of  soil  for  colon  bacilli,  while  demon- 


3  Fraenkel,  Zeit.  f .  Hyg.,  xi,  1887.  • 

4Petri,  Arb.  a.   d.  kais.  Gesundheitsamt,  vii. 

B  Firth  and  Horrocks,  Brit.  Med.  Jour.,  Sept.,  1902. 


BACTERIA   IN   THE  AIR  AND  SOIL  1015 

strating  the  presence  or  absence  of  manure  or  sewage  contamination, 
has  no  practical  value,  since  colon  bacilli  are  found  in  the  dejecta 
of  animals. 

Examination  of  specimens  of  soil  for  their  numerical  bacterial 
contents  is  extremely  unsatisfactory  because  the  bacteria  there 
found  can  hardly  ever  all  be  cultivated  together  under  one  and  the 
same  cultural  environment.  A  large  number  arc  anaerobic,  others 
again  thrive  at  low  temperatures,  while  again  another  class  may 
require  unusually  high  temperatures.  When  such  examinations  are 
made,  however,  specimens  of  the  soil  from  the  surface  layer  may  be 
taken  in  a  sterile  platinum  spoon.  When  taken  from  the  lower 
levels,  a  drill,  such  as  that  devised  by  Fraenkel,  may  be  used.  This 
consists  of  an  iron  rod  the  lower  end  of  which  is  pointed.  Just 
above  the  point  a  movable  collar  is  fitted.  This  collar  has  a  slit-like 
opening.  The  rod  beneath  the  collar  has  a  deep  longitudinal  groove 
corresponding  to  the  slit  in  the  collar.  A  flange  on  the  collar  permits 
opening  and  closing  of  the  groove  while  the  instrument  is  below  the 
ground.  The  drill  is  forced  into  the  earth  to  the  desired  depth, 
the  groove  is  opened  and  earth  is  forced  into  the  chamber  by  twist- 
ing the  rod.  In  the  same  manner  the  groove  may  be  closed.  The 
soil  obtained  in  this  way  is  taken  out  of  the  chamber  and  a  definite 
quantity,  say  one  gram,  is  dissolved  and  washed  thoroughly  in  a 
measured  volume  of  sterile  water  or  sterile  salt  solution.  Fractions 
of  this  are  then  mixed  with  the  culture  medium,  plated,  and  cul- 
tivated aerobically  or  anaerobically  as  desired. 


CHAPTER   LII 

BACTERIA  IN  WATER 

ALL  natural  waters  contain  a  more  or  less  abundant  bacterial 
flora.  This  fact,  combined  with  our  knowledge  that  the  incitants  of 
several  epidemic  diseases  and  a  number  of  minor  ailments  of  a 
diarrheal  character  are  water  borne,  gives  the  bacteriological  in- 
vestigation of  water  a  place  of  great  importance  in  hygiene.  In 
nature,  there  are  very  few  sources  of  water  supply  which  do  not 
contain  bacteria  of  one  or  another  description.  While  pathogenic 
bacteria  are  usually  not  present  except  in  those  waters  which  are 
directly  contaminated  from  human  sources,  a  thorough  understand- 
ing of  the  quantitative  and  qualitative  bacterial  contents  of  all 
natural  waters  is  necessary  in  order  that  we  may  intelligently  gather 
comparative  data  as  to  the  fitness  of  any  given  water  for  human 
consumption. 

The  gross  appearance  of  water  is  rarely,  if  ever,  an  indication 
of  its  danger.  The  turbid  waters  of  running  streams  in  sparsely 
populated  agricultural  districts  may  be  safe,  while  perfectly  clear 
well  waters  subjected  to  the  dangers  of  contamination  from  neigh- 
boring sinks  or  cess-pools  may  contain  large  numbers  of  pathogenic 
germs. 

The  diseases  which  are  known  to  be  more  directly  connected  with 
water  supply  are  typhoid  fever  and  cholera. 

Typhoid  germs  discharged  from  the  bowel  or  from  the  urine  of 
typhoid  patients  or  convalescents  may  be  carried  by  the  sewage  or 
from  the  neighboring  soil  into  a  river  or  lake  and  lead  to  infection 
of  the  population  deriving  its  drinking  water  from  this  source. 
There  are  a  great  many  investigations  on  record  in  which  severe 
typhoid  epidemics  have  been  traced  to  such  sources. 

In  the  case  of  cholera,  where  the  germs  are  discharged  from  the 
bowels  in  enormous  numbers,  conveyance  of  the  disease  by  water 
is  even  more  apparent,  and  the  discoverers  of  the  cholera  germ 
themselves,  in  their  early  work  in  Egypt  and  India,  were  able  to 
isolate  the  bacteria  from  contaminated  water  supplies. 

1016 


BACTERIA  IN  WATER  1017 

In  regard  to  the  less  clearly  understood  diarrheal  diseases,  dysen- 
tery, cholera  infantum,  etc.,  the  direct  relation  to  water  supply  has 
not  been  so  definitely  proven,  and  can  be  deduced  only  from  the 
diminution  of  such  infections  after  the  substitution  of  pure  water 
for  the  previously  used  impure  supply.  It  is  thus  seen  that  water 
bacteriology  is  one  of  the  most  important  branches  of  the  science 
of  hygiene,  and  has  led,  and  is  constantly  leading,  to  enormous 
diminution  of  the  death  rate  in  all  communities  where  an  intelligent 
study  of  the  conditions  has  been  made. 

The  bacterial  purity  of  natural  waters,  although  dependent  upon 
special  and  local  conditions  in  relation  to  possible  contamination, 
differs  widely,  according  to  the  source  from  which  such  waters  are 
derived. 

Rain  water  and  snow  water  are  usually  contaminated  with  bac- 
teria by  the  dust  which  they  gather  on  their  way  to  the  ground, 
and  are  especially  rich  in  bacteria  when  taken  during  the  first  few 
hours  of  a  rain  or  snow  storm  when  the  air  is  still  dusty  and  filled 
with  floating  particles.  During  the  later  hours  of  prolonged  storms, 
rain  water  and  snow  water  may  be  comparatively  sterile.  Miquel,1 
who  made  extensive  experiments  in  France  on  the  bacterial  contents 
of  rain  water,  found  that  in  country  districts,  where  the  air  is  less 
dusty,  rain  water  contained  an  average  of  about  4.3  bacteria  to  the 
cubic  centimeter. 

The  bacterial  counts  of  snow  water  are  usually  somewhat  higher 
than  those  of  rain. 

The  waters  of  streams,  ponds,  and  lakes  are  usually  spoken  of  as 
surface  waters,  and  these  of  all  natural  supplies  contain  the  largest 
number  of  bacteria.  In  each  case,  of  course,  the  quantitative  and 
qualitative  bacterial  flora  of  such  waters  is  intimately  dependent 
upon  the  conditions  of  the  surrounding  country,  the  density  of  the 
population,  and  the  relation  of  these  waters  to  sewage.  It  is  also, 
and  to  no  less  important  degree,  dependent  upon  weather  conditions, 
the  influence  of  light  and  temperature,  and  the  food  supply  contained 
within  the  waters  in  the  form  of  decayed  vegetation.  In  all  such 
surface  waters  there  is  constantly  going  on  a  process  of  self-purifica- 
tion. The  chief  factor  in  this  process  is  sedimentation.  In  stagnant 
ponds  and  lakes  with  but  sluggish  currents  there  is  a  constant 
sedimentation  of  the  heavier  particles,  which  gradually  but  steadily 

1  Miquel,  Revue  d'hyg.,  viii,  1886. 


1018  BACTERIA  IN  AIR,  SOIL,   WATER,  AND  MILK 

leads  to  a  diminution  of  the  number  of  bacteria  in  the  upper  layers 
of  the  water.  In  rivers  where  sedimentation  is  to  a  certain  extent 
prevented  by  rapidity  of  Current,  the  effectiveness  of  such  sedimen- 
tation is,  of  course,  entirely  dependent  upon  the  speed  of  the  current. 

The  influence  of  light  in  purifying  surface  waters  is  important 
chiefly  in  ponds,  lakes,  and  sheets  of  water  which  expose  a  large 
surface  to  the  sunlight,  and  where  the  surroundings  are  such  that 
the  sun  has  free  access  throughout  the  day.  According  to  the 
researches  of  Buchner,2  the  bactericidal  effects  of  light  penetrate 
through  water  to  a  depth  of  three  feet. 

The  effects  of  temperature  in  purifying  surface  waters  under 
natural  conditions  are  probably  not  great.  There  is,  however,  a 
general  tendency  toward  diminution  of  the  bacterial  flora  as  the 
temperature  of  such  waters  becomes  lower. 

The  presence  of  protozoa  in  natural  waters  as  purifying  agents 
has  recently  been  emphasized  by  Huntemiiller,3  who  claims  that 
these  organisms  by  phagocytosis  greatly  diminish  the  number  of 
bacteria  in  any  given  body  of  water.  It  is  self-evident  that  the 
number  of  bacteria  in  any  of  these  waters  is  never  constant,  since 
all  factors  which  tend  to  a  diminution  or  increase  in  volume,  such 
as  drying  up  of  tributary  streams  or  the  occurrence  of  heavy  rains, 
would  lead  to  differences  of  dilution  which  would  materially  change 
numerical  bacterial  estimations.  The  influence  of  rains,  furthermore, 
may  be  a  twofold  one.  On  the  one  hand,  heavy  rain-falls,  by  wash- 
ing a  large  amount  of  dirt  into  the  rivers  and  lakes  from  the  sur- 
rounding land,  have  a  tendency  to  increase  the  bacterial  flora.  This 
influence  would  be  especially  marked  in  all  bodies  of  water  which 
are  surrounded  by  cultivated  land  where  manured  fields  and  graz- 
ing-meadows  supply  a  plentiful  source  of  bacteria.  On  the  other 
hand,  in  regions  where  arid,  uninhabited  lands  surround  any  given 
river  or  lake,  the  rain  would  carry  with  it  very  little  living  con- 
tamination and  would  act  chiefly  as  a  diluent  and  diminish  the 
actual  proportion  of  bacteria  in  the  water. 

Another  and  extremely  important  source  of  water  supply  is  that 
spoken  of  technically  as  "ground  water/'  The  "ground  waters" 
include  the  shallow  wells  employed  in  the  country  districts,  springs, 
and  deep  or  artesian  wells.  The  shallow  wells  that  form  the  water 


2  Buchner,  Arch.  f.  Hyg.,  xvii,  1895. 
8  HuntemiiUer,  Arch.  f.  Hyg.,  liv,  1905. 


BACTERIA   IN   WATER  1019 

supply  for  a  large  proportion  of  farms  in  the  eastern  United  States 
are  usually  very  rich  in  bacteria  and  are  by  no  means  to  be  regarded 
as  safe  sources,  except  in  cases  where  great  care  is  observed  as 
to  cleanliness  of  the  surroundings.  In  such  wells  the  filtration  of 
the  water  entering  the  well  may  be  subject  to  great  variation  accord- 
ing to  the  geological  conditions  of  the  surrounding  ground.  The 
proximity  of  barns  and  sinks  may  lead  to  dangerous  contamination 
of  such  waters. 

Examinations  by  various  bacteriologists  have  shown  that  such 
wells  frequently  contain  as  many  as  five  hundred  bacteria  to  the 
cubic  centimeter. 

Perennial  spring  waters  are  usually  pure.  Examinations  by  the 
Massachusetts  State  Board  of  Health4  in  1901  showed  an  average 
of  about  forty  bacteria  per  cubic  centimeter.  As  sources  of  water 
supply  for  general  consumption,  however,  springs  can  hardly  be 
very  important  because  of  the  insignificant  quantities  usually  derived 
from  them. 

Of  much  greater  practical  importance  are  deep  artesian  wells, 
which,  under  ordinary  conditions,  are  largely  free  from  bacterial 
contamination. 

Quantitative  Estimations  of  Bacteria. — The  quantitative  estima- 
tion of  bacteria  in  water  is  of  necessity  inexact,  because  of  the 
difficulty  of  always  securing  fair  average  samples  from  any  large 
body  of  water,  and  because  of  the  large  variations  in  cultural  re- 
quirements of  the  flora  present  in  them.  All  these  methods  depend 
upon  colony  enumeration  in  plates  of  agar  or  gelatin,  preferably 
of  both.  For  the  sake  of  gaining  some  basis  of  comparison  for 
results  which,  at  best,  can  never  be  entirely  accurate,  an  attempt 
has  been  made  by  the  American  Public  Health  Association5  to 
standardize  the  methods  of  analysis. 

Water  for  analysis  should  always  be  collected  in  clean,  sterile 
bottles,  preferably  holding  more  than  100  c.c.  If  w^ater  is  to  be 
taken  from  a  running  faucet  or  a  well  supplied  with  piping,  it  is 
important  that  it  should  be  allowed  to  run  for  some  time  before 
the  sample  is  taken,  in  order  that  any  change  in  bacterial  content 
occurring  inside  of  the  pipes  may  be  excluded.  It  is  obvious  that 
in  water  pipes  through  which  the  flow  is  not  constant,  bacteria  may 

4  Mass.  State  Bd.  of  Health,  33d  Annual  Eeport  for  1901. 

5  Fuller,  Trans.  Amer.  Public  Health  Assn.,  xxvii,   1902.     Eeport  of  Com.  on 
Standard  Methods  of  Water  Analysis,  Jour.  Inf.  Dis.,  Suppl.  1,  1905. 


1020  BACTERIA   IN   AIR,   SOIL,   WATER,   AND   MILK 

find  favorable  conditions  for  growth  and  such  a  sample  would  not 
represent  fairly  the  supply  to  be  tested.  When  the  water  is  taken 
from  a  pond,  lake,  or  cess-pool,  the  bottle  may  be  lowered  into  the 
water  by  means  of  a  weight,  or  may  be  plunged  in  with  the  hand, 
great  care  being  exercised  not  to  permit  contamination  from  the 
fingers  to  occur. 

After  the  water  has  been  collected  it  is  important  to  plate  it 
before  the  bacteria  in  it  have  a  chance  to  increase.  The  changes 
taking  place  during  transportation,  even  when  packing  in  ice  has 
been  resorted  to,  have  been  found  by  Jordan  and  Irons6  to  be 
considerable.  It  is  imperative,  therefore,  that  plating  of  the  water, 
if  possible,  shall  not  be  delayed  for  longer  than  one  or  two  hours 
after  collection. 

Bacteriological  Examination  of  Water. — In  describing  the 
methods  of  bacteriological  examinations  of  water,  we  adhere  strictly 
to  the  recommendations  of  the  Committee  on  Standard  Methods  of 
the  American  Public  Health  Association,7  taking  the  following 
paragraphs  with  slight  changes  from  their  report  of  1915 : 

It  should  be  remembered  that  quantitative  estimations  of  bacteria 
in  water  are  of  most  value  when  repeatedly  done  and  a  " normal" 
for  the  particular  water  supply  has  been  established,  so  that  devia- 
tion from  this  "normal"  can  be  easily  recognized.  Single  isolated 
determinations  may  easily  lead  to  error. 

The  following  paragraphs  are  taken  without  change  from  the 
Public  Health  Association 's  report : 

"Since  gelatine  does  not  give  the  total  number  of  bacteria  in  the  water, 
the  committee  has  thought  it  wise  to  use  agar  incubated  at  37°  C.  as  a 
standard  medium.  This  admits  of  counts  in  one  day  instead  of  two,  and 
gives  results  on  the  kind  of  bacteria  growing  at  blood  temperature  and 
therefore  more  nearly  related  to  pathogenic  types'. 

"Media. — The  standard  medium  for  determining  the  number  of  bacteria 
in  water  shall  be  nutrient  agar.  All  variations  from  this  medium  shall  be 
considered  special  media.  If  any  medium  other  than  standard  agar  is  used, 
this  fact  shall  be  stated  in  the  report. 

"For  general  work  the  standard  reaction  shall  be  +1  per  cent,  but  for 
long  continued  work  upon  water  from  the  same  source  the  optimum  reaction 
shall  be  ascertained  by  experiment  and  thereafter  adhered  to.  If  the  reaction 
used,  however,  is  different  from  the  standard,  it  shall  be  so  stated  in  the 
report. 

•  Jordan  and  Irons,  Eeports  of  the  Amer.  Pub.  Health  Assn.,  xxv,  1889. 
7Amer.  P.  H.  A.,  Stand.  Meth.  Exam.  Water  and  Sewage,  1915. 


BACTERIA  IN   WATER 


1021 


"Procedure. — Shake  at  least  twenty-five  times  the  bottle  which  contains 
the  sample.  Withdraw  1  c.c.  of  the  sample  with  a  sterilized  pipette  and 
deliver  it  into  a  sterilized  Petri  dish,  10  cm.  in  diameter.  If  there  be  reason 
to  suspect  that  the  number  of  bacteria  is  more  than  200  per  c.c.,  mix  1  c.c. 
of  the  sample  with  9  c.c.  of  sterilized  tap  or  distilled  water.  Shake  twenty-five 
times  and  measure  1  c.c.  of  the  diluted  sample  into  a  Petri  dish.  If  a  higher 
dilution  be  required  proceed  in  the  same  manner,  e.g.,  1  c.c.  of  the  sample  to 
99  c.c.  of  sterilized  water,  or  1  c.c.  of  the  once  diluted  sample  to  9  c.c.  of 
sterilized  water,  and  so  on.  In  the  case  of  an  unknown  water  or  a  sewage 
it  is  advisable  to  use  several  dilutions  for  the  same  sample.  To  the  liquid 
in  the  Petri  dish  add  10  c.c.  of  standard  agar  at  a  temperature  of  about 
40°  C.  Mix  the  medium  and  water  thoroughly  by  tipping  the  dish  back  and 
forth,  and  spread  the  contents  uniformly  over  the  bottom  of  the  plate.  Allow 
the  agar  to  cool  rapidly  on  a  horizontal  surface  and  transfer  to  the  37°  C. 
incubator  as  soon  as  it  is  hard.  Incubate  the  culture  for  twenty-four  hours 
at  a  temperature  of  37°  C.  in  a  dark,  well-ventilated  incubator  where  the 
atmosphere  is  practically  saturated  with  moisture.8  After  the  period  of 
incubation  place  the  Petri  dish  on  a  glass  plate  suitably  ruled  and  count 
the  colonies  with  the  aid  of  a  lens  which  magnifies  at  least  five  diameters. 
So  far  as  practicable  the  number  of  colonies  upon  the  plate  shall  not  be 
allowed  to  exceed  200.  The  whole  number  of  colonies  upon  the  plate  shall 
be  counted,  the  practice  of  counting  a  fractional  part  being  resorted  to  only 
in  case  of  necessity. 

"It  will  be  found  advantageous  to  use  Petri  dishes  with  porous  earthen- 
ware covers  in  order  to  avoid  the  spreading  of  colonies  by  the  water  of 
condensation.9 

"Expression  of  Results. — In  order  to  avoid  fictitious  accuracy  and  yet  to 
express  the  numerical  results  by  a  method  consistent  with  the  precision  of  the 
work  the  rules  given  below  shall  be  followed : 


''Numbers  of  Bacteria  per  c.c. 


From 


1  to 


50  Recorded  as  found 


51  "  100 

101  "  250 

251  "  500 

501  "  1,000 

1,001  "  10,000 

10,001  "  50,000 

50,001  "  100,000 

100,001  "  500,000 

500,001  "  1,000,000 

1,000,001  "  10,000,000 


to  the  nearest 


5 
10 
25 

50 

100 

500 

1,000 

10,000 

50,000 

100,000" 


Qualitative  Bacterial  Analyses  of  Water. — Of  far  greater  im- 
portance than  quantitative  analysis  is  the  isolation  of  bacteria  either 

8  Whipple,  Tech.  Quar.,  1899,  12,  p.  276. 

9  Hill,  Jour.  Med.  Res.,  1904,  N.-S.,  8,  p.  93. 


1022  BACTERIA  IN   AIR,   SOIL,   WATER,   AND   MILK 

distinctly  pathogenic,  such  as  the  cholera  spirillum  and  the  typhoid 
bacillus,  or  of  other  species  probably  emanating  from  contaminating 
sources,  such  as  a  B.  coli.  Unfortunately  there  are  no  reliable 
methods  by  which  typhoid  and  cholera  germs  can  be  isolated  from 
water  with  any  degree  of  regularity  or  certainty.  Although  fre- 
quently the  isolation  of  such  organisms  is  possible,  a  negative  result 
in  these  cases  is  by  no  means  eliminative  of  their  presence. 

The  isolation  of  typhoid  bacilli  from  water  is  very  difficult,  chiefly 
because  of  the  great  dilution  which  contaminations  undergo  upon 
entering  any  large  body  of  water.  The  difficulty  of  isolating  typhoid 
bacilli,  even  from  the  stools  of  infected  patients,  makes  it  clear  that 
such  difficulties  are  enhanced  when  a  considerable  dilution  of  the 
excreta  takes  place.  Furthermore,  water  is  by  no  means  a  favorable 
medium  for  the  typhoid  bacillus.  Russell  and  Fuller10  have  shown 
that  typhoid  bacilli  may  die  in  water  within  five  days,  and  it  is 
unquestionable  that  the  rate  of  increase  of  these  bacteria  is  by  no 
means  equal  to  that  of  many  other  microorganisms  for  which  pol- 
luted water  at  the  temperature  encountered  in  streams  and  lakes 
forms  a  much  more  favorable  medium. 

It  is  thus  clear  that  even  in  infected  waters  the  number  of  typhoid 
bacilli  encountered  can  never  be  very  great.11 

A  large  number  of  methods  for  the  isolation  of  the  typhoid 
bacillus  from  water  have  been  devised.  Most  of  the  media  used 
are  identical  with  those  employed  for  the  isolation  of  these  bacteria 
from  the  stools.  These  media  have  been  discussed  in  the  chapter 
dealing  with  the  typhoid  bacillus.  Success  is  rendered  more  likely 
if  10  c.c.  of  the  water  is  first  planted  into  lactose-bile  in  fermentation 
tubes  holding  40  c.c.  After  forty-eight  hours  at  37.5°  there  will 
be  an  enrichment  of  typhoid  bacilli  which  can  be  then  isolated  by 
plating  in  the  usual  manner  on  Endo  's  medium,  Conradi  Drigulski  or 
any  of  the  other  usual  differential  media. 

A  method  which  has  proved  useful  in  the  hands  of  Adami  and 
Chapin12  is  one  which  depends  upon  the  phenomenon  of  agglutina- 
tion. They  attempt  to  agglutinate  the  bacilli  out  of  liter  samples 
of  water  by  adding  powerful  agglutinating  serum. 

Vallet  and  others  have  attempted  to  precipitate  typhoid  bacilli 
out  of  water  by  chemical  means.  To  two  liters  of  water  add  20  c.c. 


™Eussell  and  Futter,  Jour.  Inf.  Dis.,  Suppl.  2,  1908. 

11  Laws  and  Anderson,  Rep.  of  Med.  Officer,  London  County  Council,  1894. 

J2  Adami  and  Chapin,  Jour.  Med.  Res.,  xl,  1904. 


BACTERIA   IN   WATER  1023 

of  a  7.75  per  cent  solution  of  sodium  hyposulphite  and  20  c.c.  of  a 
10  per  cent  solution  of  lead  nitrate.  When  the  precipitate  has  settled, 
the  clear  supernatant  fluid  is  decanted  and  the  precipitate  dissolved 
in  a  saturated  sodium  hyposulphite  solution.  This  clear  solution  is 
then  plated.  Willson13  has  modified  this  method  by  adding  to  the 
water  0.5  gm.  of  alum  to  each  liter.  The  supernatant  fluid  is  removed 
and  the  precipitate  plated. 

The  isolation  of  the  vibrio  of  cholera  is  less  difficult  than  that 
of  B.  typhosus,  primarily  because  of  the  much  greater  numbers  of 
these  microorganisms  discharged  into  sewage.  The  number  of 
cholera  spirilla  in  the  excreta  of  cholera  patients  is  enormously 
higher  than  is  that  of  B.  typhosus  in  the  stools  of  typhoid-fever 
patients.  It  is  not  infrequent,  therefore,  that  the  source  of  a  cholera 
infection  may  be  directly  traced  to  the  water  supply.  Koch,1*  the 
discoverer  of  the  cholera  vibrio,  has  indicated  a  method  which  has 
frequently  found  successful  application. 

To  100  c.c.  of  the  infected  water  are  added  one  per  cent  of 
pepton  and  one  per  cent  of  salt.  This  mixture  is  then  incubated  at 
37.5°  C.,  and  after  ten,  fifteen,  and  twenty  hours,  specimens  from 
the  upper  layers  are  examined  microscopically  and  are  plated.  The 
scum  from  the  surface  of  such  a  medium  may  be  plated  on  the 
starch  agar  of  Stokes  and  Haechtel,15  on  which  colonies  of  intestinal 
spirilla  will  appear  pink  and  spreading. 

Because  of  the  great  difficulties  outlined  above  in  isolating  specific 
pathogenic  germs  from  polluted  waters,  bacteriologists  have  at- 
tempted to  form  an  approximate  estimation  of  pollution  by  the 
detection  of  other  microorganisms  which  form  the  predominating 
flora  of  sewage.  Chief  among  these  is  B.  coli.  The  isolation  and 
numerical  estimation  of  B.  coli  in  polluted  water  has  been  for  a 
long  time  one  of  the  criteria  of  pollution.  This  so-called  colon  test, 
however,  should  always  be  approached  with  conservatism  and  should 
never  be  carried  out  qualitatively  only.  Careful  quantitative  es- 
timation should  be  made  in  every  case. 

B.  coli  in  water  is  by  no  means  always  the  result  of  human  con- 
tamination, since  this  bacillus  is  found  in  great  abundance  in  the 


13  Willson,  Jour,  of  Hyg.,  v,  1905. 

"Koch,  Zeit.  f.  Hyg.,  xiv,  1893. 

15  Stokes  and  Haechtel,  see  Eeport  1915  A.  P.  H.  A.,  on  Water  Analysis.  The 
medium  is  an  agar  with  5.5  grams  agar,  5.0  meat  extract,  10  Pepton  and  8.5  NaCl 
to  liter  to  which  is  added  10  grams  of  soluble  starch. 


1024  BACTERIA  IN  AIR,  SOIL,   WATER,   AND   MILK 

intestines  of  domestic  animals.  According  to  Poujol,  B.  coli  does 
not  even  always  point  to  fecal  contamination,  since  this  author  was 
able  to  find  the  bacillus  in  the  water  of  a  number  of  wells  where 
no  possible  contamination  of  any  sort  could  be  traced.  Prescott18 
explains  this,  as  well  as  similar  cases,  by  the  fact  that  organisms 
of  the  colon  group  may  occasionally  be  parasitic  upon  plants. 

The  opinions  of  hygienists  are  widely  at  variance  as  to  the  value 
of  the  colon  test.  While  the  discovery  of  isolated  bacilli  of  the  colon 
group  may  therefore  be  of  little  value,  it  is  nevertheless  safe  to 
follow  the  opinion  of  Houston,17  who  states  that  the  discovery  of 
B.  coli  in  considerable  numbers  invariably  points  to  sewage  pollu- 
tion, and  that  the  absolute  absence  of  B.  coli  is,  as  a  rule,  reliable 
evidence  of  purity. 

Rosenau  states  that  a  ground  water  should  be  condemned  even 
if  only  a  few  colon  bacilli  are  found,  for,  as  he  put  it,  '  *  these  bacteria 
have  no  business  in  a  soil-filtered  and  properly  protected  well  or 
spring-water."  Surface  waters,  however,  may  easily  contain  a  few 
colon  bacilli  without  necessarily  having  been  exposed  to  contamina- 
tion by  human  forces.  The  limit  of  safety,  Rosenau  states,  is  one 
colon  bacillus  per  c.c.  If  more  are  present  the  water  should  be 
regarded  as  suspicious.  If  more  than  10  per  c.c.  are  found  the 
water  must  be  regarded  as  dangerous  and  unqualifiedly  condemned. 

Presumptive  Colon  Tests. — For  this  purpose,  a  large  number  of 
methods  have  been  devised.  In  examining  sewage  or  other  polluted 
waters  in  which  the  number  of  colon  bacilli  is  comparatively  large, 
the  direct  use  of  lactose  litmus  agar  plates  yields  excellent  results. 
The  method  advised  by  the  American  Public  Health  Association  is 
as  follows : 

"Add  the  quantities  of  water  or  sewage  to  be  tested  to  fermentation  tubes 
holding  at  least  forty  cubic  centimeters  Q£  lactose  bile,18  incubate  at  37°  C. 
and  note  the  production  of  gas.  The  standard  time  for  observing  gas  produc- 
tion is  forty-eight  hours.  Small  numbers  of  somewhat  attenuated  B.  coli 
may  require  three  days  to  form  gas.  Attenuated  B.  coli  does  not  represent 
recent  contamination  and  all  B.  coli  not  attenuated  grows  readily  in  lactose 
bile.  No  other  organism  except  B.  Welchii  gives  such  a  test  in  lactose  bile. 
B.  Welchii  is  of  rather  rare  occurrence  in  water,  is  of  fecal  origin,  is  almost 
invariably  accompanied  by  B.  coli,  and  the  sanitary  significance  is  the  same. 

"Prescott,  Science,  xv,  1903. 

17  Houston,  Kep.  Medical  Officer,  Local  Gov.  Board,  London,  1900. 

18  Prescott,  Science,  xvi,  1902. 


BACTERIA  IN  WATER  1025 

"A  comparison  of  the  positive  results  obtained  in  the  various  dilutions 
of  the  water  or  sewage  planted  into  the  lactose  bile  gives  a  good  idea  of 
the  relative  amount  of  contamination  in  the  various  samples  examined. 

"Quantities  of  Water  Tested. — For  ordinary  waters,  0.1,  1.0  and  10.0  c.c. 
shall  be  used  for  the  colon  test.  For  sewage  and  highly  polluted  surface 
waters,  smaller  quantities  shall  be  used;  and  for  ground  waters,  filtered 
waters,  etc.,  the  quantities  shall  be  larger,  if  necessary  to  obtain  positive 
results.  The  quantities  shall  vary  preferably  in  the  tenfold  manner  in- 
dicated. Single  tests  with  quantities  which  give  ordinarily  a  positive  result 
or  ordinarily  a  negative  result  are  in  themselves  of  but  little  account  for 
quantitative  determinations.  The  range  in  quantities  studied  shall  be  sufficient 
to  allow  the  quantities  needed  for  both  a  positive  and  a  negative  result  to 
be  recorded  for  each  sample.  When  this  is  done,  the  results  of  several  tests 
allow  an  approximate  estimate  of  the  number  of  B.  coli  per  c.c." 

The  identification  of  colon  bacilli  so  obtained  should  then  be 
undertaken.  The  following  table,  again  taken  from  the  report  of 
the  A.  P.  H.  A.,  will  be  of  help : 


1026  BACTERIA  IN  AIR,   SOIL,   WATER,  AND  MILK 

+  +  I  +  I 


I    | 

}* 'a 

PQ  ~ 


£ 


PQ 


+ 


PQ 


11 

§  a 


ffl'M 


• 


—  i  a  — 


+  +  +  i  i 

.1 

22§§§ 

+  +  +  i  i  +  i     i 

A.  r^H 

+  +  +  +  +    |      | 

'3  ~   ^   w   v. 
I 

•<i3<^<3CQPQCjQ          d 
+    I    ++  | 

02  O> 

i"J     ! 
++ 1 '     i 

'3 «  >.  « 

•<1  PQ  O  Q 

+  +    I    +    I  | 

+  +  +    I     I  | 

'S  ~   ^   ^   ^ 

I 


CHAPTER   LIII 

BAGTEEIA  IN  MILK  AND  MILK  PRODUCTS,  BACTERIA  IN  THE 

INDUSTRIES 

BACTERIA    IN   MILK 

THE  universal  use  of  cows'  milk  as  a  food,  especially  for  the 
nourishment  of  infants,  has  necessitated  its  close  study  by  bac- 
teriologists and  hygienists.  It  furnishes  an  excellent  culture  medium 
for  bacteria  and  is,  therefore,  pre-eminently  fitted  to  convey  the 
germs  of  infectious  disease.  The  many  changes  which  take  place 
in  milk,  furthermore,  and  which  add  or  detract  from  its  nutritive 
value,  are  due  largely  to  bacterial  growth  and  have  been  elucidated 
by  bacteriological  methods. 

Within  the  udder  of  the  healthy  cow,  milk  is  sterile.  If  pyogenic 
or  systemic  diseases  of  bacterial  origin  exist  in  the  cow,  the  milk 
may,  under  certain  circumstances,  be  infected  even  within  the  mam- 
mary glands.  In  the  milk  ducts  and  in  the  teats,  however,  even 
in  perfectly  healthy  animals,  a  certain  number  of  bacteria  may  be 
found.  For  this  reason,  even  when  all  precautionary  measures  are 
followed,  the  milk  as  received  in  the  pail  is  usually  contaminated. 
As  a  matter  of  fact,  the  anatomical  location  of  the  udder  and  the 
mechanical  difficulties  of  milking  make  it  practically  impossible  to 
collect  milk  under  absolutely  aseptic  conditions,  and,  under  the  best 
circumstances,  from  100  to  500  microorganisms  per  c.c.  may  usually 
be  found  in  freshly  taken  milk.  Withdrawn  under  conditions  of 
ordinary  cleanliness,  the  bacterial  contents  of  milk  are  considerably 
higher  than  this.  After  the  process  of  milking,  in  spite  of  all  prac- 
ticable precautions,  the  chances  for  the  contamination  of  milk  are 
considerable ;  but  even  could  these  be  eliminated,  the  bacterial  con- 
tents of  a  given  sample  would  ultimately  rapidly  increase  because 
of  the  rich  culture  medium  which  the  milk  provides  for  bacteria. 
Whether  large  increases  shall  take  place  or  not  depends,  in  the 
first  place,  upon  the  temperature  at  which  milk  is  kept,  and,  in 
the  second  place,  upon  the  length  of  time  which  intervenes  before 

1027 


1028  BACTERIA  IN   AIR,   SOIL,   WATER,  AND   MILK 

its  consumption.  Though  fresh  milk  possesses  slight  bactericidal 
powers,1  these  are  by  no  means  sufficient  to  be  of  practical  im- 
portance in  the  inhibition  of  bacterial  growth.  Kept  at  or  about 
freezing-point,  the  bacterial  contents  of  milk  do  not  appreciably 
increase.  At  higher  temperatures,  however,  a  rapid  propagation  of 
bacteria  takes  place  which,  especially  during  the  summer  months, 
speedily  leads  to  enormous  numbers.  In  a  case  reported  by  Park,2 
where  milk,  containing  at  the  first  examination  30,000  microorgan- 
isms per  cubic  centimeter,  was  kept  at  30°  C.  (86°  F.)  for  twenty- 
four  hours,  the  count  at  the  end  of  this  time  yielded  fourteen 
billions  of  bacteria  for  the  same  quantity. 

It  is  of  much  importance,  therefore,  that  the  cleanliness  of  dairies, 
of  cattle,  and  in  the  handling  of  milk  should  be  reinforced  by  the 
utmost  care  in  chilling  and  icing  during  shipment  and  before  sale. 

Because  of  its  great  importance,  especially  for  the  health  of  the 
children  in  large  cities  during  the  summer  months,  the  milk  question 
has,  of  recent  years,  received  much  attention  from  health  officers. 
In  the  city  of  New  York,  the  question  has  been  made  the  subject 
of  many  careful  studies  by  Park3  and  his  associates.  Commissions, 
working  in  Chicago,4  Boston,5  and  other  large  towns,  have  placed 
the  sale  of  milk  under  more  or  less  exact  bacteriological  supervision. 
Park  has  determined  that  the  milk,  as  sold  in  New  York  stores 
during  the  cold  weather,  not  infrequently  averages  seven  hundred 
and  fifty  thousand  bacteria  per  cubic  centimeter;  during  the  hot 
summer  months,  the  bacterial  contents  of  similar  milk  not  infre- 
quently average  one  million  and  more,  for  the  same  quantity.6  In 
consequence  of  these  and  other  researches,  large  dairies  have  intro- 
duced bacteriological  precautions  into  their  method  of  milk  produc- 
tion. They  have  attempted  the  reduction  of  the  bacterial  contents 
of  milk  by  scrupulous  cleanliness  of  the  barns  and  of  the  udders 
and  teats  of  the  cow,  by  the  elimination  of  diseased  cattle,  by 
sterilization  of  the  vessels  in  which  the  milk  is  received,  and  of  the 
hands  of  the  milker;  also  by  the  immediate  filtering  and  cooling 
of  the  milk  and  the  packing  of  the  milk  cans  in  ice,  where  they 

1  Eosenau  and  McCoy,  Jour.  Med.  Ees.,  18,  1908. 

*Park,  W.  H.,  "Pathogenic  Bacteria,"  New  York,  1905,  p.  463. 

3  Park,  Jour,  of  Hygiene,  1,  1901. 

4  Jordan  and  Hcinemann,  Rep.  of  the  Civic  Federation  of  Chicago,  1904. 

5  Sedgwick  and  Batchelder,  Bost.  Med.  and  Surg.  Jour.,  126,  1892. 
*Escherich,  Fort.  d.  Medizin,  16  and  17,  1885. 


BACTERIA  IN  MILK  1029 

remain  until  delivered  to  the  consumer.  In  consequence  of  such 
measures,  it  is  possible  for  cities  to  be  supplied  with  milk  containing 
no  more,  and  often  less,  than  fifty  thousand  bacteria  to  the  cubic 
centimeter.  A  standard  of  cleanliness  has  been  established  in  various 
towns  by  the  introduction  of  the  so-called  "certified  milk,"  which, 
by  the  New  York  Milk  Commission,  is  required  to  contain  no  more 
than  thirty  thousand  bacteria  per  cubic  centimeter.  Great  stress 
is  laid  upon  such  numerical  counts  simply  in  that  they  are  ap- 
proximate estimates  of  cleanliness.  Most  of  the  bacteria,  however, 
contained  in  milk  are  non-pathogenic,  and  numbers  much  larger 
than  the  maximum  required  for  certified  milk  may  be  present  with- 
out actual  disease  or  harm  following  its  consumption. 

The  various  species  of  bacteria  which  may  be  found  in  milk 
include  almost  all  known  varieties.  Whether  there  are  special, 
so-called  milk  bacteria  or  not  is  a  question  about  which  investigators 
have  expressed  widely  differing  opinions.  It  is  probable  that  many 
of  the  species,  formerly  regarded  as  specifically  belonging  to  milk, 
are  there  simply  by  virtue  of  their  inhabitual  presence  in  fodder, 
straw,  or  bedding,  or  upon  cattle.  It  is  likely,  furthermore,  that 
some  of  these  species  are  found  with  great  regularity  because  of  their 
power  to  outgrow  other  species  under  the  cultural  conditions  offered 
them  in  milk. 

Under  normal  conditions,  milk  always  undergoes  a  process  which 
is  popularly  known  as  souring  and  curdling.  This  is  due  to  the 
formation  of  lactic  acid  from  the  milk  sugar  and  is  the  result  of 
the  enzymatic  activities  of  several  varieties  of  bacteria  commonly 
found  in  milk.  Most  common  among  these  bacteria  is  the  so-called 
Bacillus  lactis  aerogenes,  an  encapsulated  bacillus  closely  related 
to  the  colon- bacillus  group.  (See  page  637.)  The  transformation 
of  the  lactose  into  lactic  acid  may  occur  either  directly  by  hydrolytic 
cleavage : 

C12  H220U  +  H20  =  4  C3  H6  0,j 
or  indirectly  through  a  monosaccharid, 

C12  H22  Olt  +  H2  O  =  2  G6  H12  06  =  4  C3  H6  O8. 

Other  microorganisms  which  may  cause  lactic-acid  fermentation  in 
milk  are  the  so-called  Streptococcus  lacticus,  the  common  pyogenic 
streptococcus,  the  Staphylococcus  aureus,  Bacillus  coli  communis  and 
communior,  and  many  other  species.  Most  commonly  concerned  in 


1030  BACTERIA  IN  AIR,  SOIL,  WATER,  AND  MILK 

lactic-acid  production,  however,  according  to  Heinemann,7  are  the 
two  first-mentioned,  Bacillus  lactis  aerogenes  and  Streptococcus 
lacticus.  The  secret  of  the  regularity  with  which  some  of  these 
bacteria  are  present  in  sour  milk  is  probably  found  in  the  ability 
of  these  varieties  to  withstand  a  much  higher  degree  of  acidity 
of  the  culture  medium  than  other  species.  In  consequence,  they  are 
able  to  persist  and  develop  when  cultural  conditions  are  absolutely 
unsuited  to  other  bacteria. 

Consequent  upon  acidification  of  the  milk  by  lactic-acid  forma- 
tion, there  is  coagulation  of  casein.  Casein  precipitation,  however, 
may  also  be  due  to  a  non-acid  coagulation  caused  by  a  bacterial 
ferment.  Casein  precipitated  in  this  way  may  be  redissolved  by 
a  bacterial  trypsin  or  casease,  produced  by  the  same  or  other  bac- 
teria, the  milk  again  becoming  entirely  liquid,  transparent,  and  of 
a  yellowish  color.  The  casein  precipitated  by  lactic-acid  formation, 
however,  is  never  thus  redissolved,  because  the  high  acidity  does 
not  permit  the  proteolytic  ferments  to  act.8 

Butyric-acid  fermentation  in  milk,  a  common  phenomenon,  is  also 
an  evidence  of  bacterial  growth.  As  a  rule,  it  is  produced  by  the 
anaerobic  bacteria,  and  is  a  process  developing  much  more  slowly 
than  other  fermentations.  A  large  number  of  bacteria  have  been 
described  which  are  capable  of  producing  such  changes,  the  chemical 
process  by  which  they  are  produced  being,  as  yet,  not  entirely 
understood.  It  is  probable  that  the  process  takes  place  after  hydrol- 
ysis of  the  dissaccharid  somewhat  according  to  the  following 
formula : 

C«  H12  Q6  —  C4  H8  O2  +  2  CO2  -f-  2  H2. 

Special  bacteria  have  been  described  in  connection  with  this  form 
of  milk  fermentation,9  most  of  them  non-pathogenic.  It  is  unques- 
tionable, however,  that  many  of  the  well-known  pathogenic  bacteria, 
such  as  Bacillus  aerogenes  capsulatus,  Bacillus  cedematis  maligni, 
possess  the  power  of  similar  butyric-acid  formation.  While  less 
commonly  observed  in  milk,  because  milk  is  rarely  kept  long  enough 
to  permit  of  the  action  or  development  of  these  enzymes,  the  butyric- 
acid  fermentation  is  of  importance  in  connection  with  butter,  where 
it  is  one  of  the  causes  producing  rancidity. 


7  Heinemann,  Jour,  of  Inf.  Dig.,  3,  1906. 

6  Conn,  Exper.  Stat.  Rep.,  1892. 

9  Schattenfroh  und  Grasberger,  Arch.  f.  Hyg.,  37,  1900. 


BACTERIA   IN    MILK  1031 

Alcoholic  fermentation  may  take  place  in  milk  as  a  result  of 
the  activities  of  certain  yeasts.  Upon  the  occurrence  of  such  fer- 
mentations depends  the  production  of  kefyr,  koumys,  and  other 
beverages  which  have  been  in  common  use  for  many  years,  especially 
in  the  region  of  the  Caucasus.  The  characteristic  quality  of  these 
beverages  is  contributed  by  the  feeble  alcoholic  fermentation  pro- 
duced by  members  of  the  saccharomyces  group,  but  side  by  side 
with  this  process  lactic-acid  formation  also  takes  place.  Beijerinck,10 
who  has  carefully  studied  the  so-called  kefyr  seeds,  used  for  the 
production  of  kefyr  in  the  East,  has  isolated  from  them  a  form  of 
yeast  similar  in  many  respects  to  the  ordinary  beer  yeast,  and  a 
large  bacillus  to  which  he  attributes  the  lactic-acid  formation. 

Occasional  but  uncommon  changes  which  occur  in  milk  lead  to 
the  formation  of  the  so-called  " slimy  milk,"  yellow  and  green  milk, 
and  bitter  milk.  These  may  be  due  to  a  number  of  bacteria.  A 
microorganism  which  is  commonly  found  in  connection  with  the 
slimy  changes  in  milk  is  the  so-called  Bacillus  lactis  viscosus.  Ac- 
cording to  the  researches  of  Ward,11  this  microorganism  is  frequently 
derived  from  water  and  it  is  the  water  supply  which  should  attract 
attention  whenever  such  trouble  occurs  in  dairies. 

The  so-called  blue,  green,  and  yellow  changes  are  usually  due  to 
chromogenic  bacteria,  such  as  Bacillus  cyanogenes,  Bacillus  prodigio- 
sus,  and  others. 

'  *  Bitter  milk, ' '  a  condition  which  has  occasionally  been  observed 
epidemically,  is  also  the  consequence  of  the  growth  of  microorgan- 
isms. Conn,12  in  1891,  isolated  from  a  specimen  of  bitter  cream  a 
diplococcus  which  occasionally  forms  chains  and  which  in  sterilized 
milk  develops  rapidly,  producing  an  extremely  bitter  taste.  The 
organism  of  Conn  differs  from  a  similar  diplococcus  described  by 
Wagmann13  in  that  it  possesses  the  ability  of  producing  butyric  acid. 

Milk  in  Relation  to  Infectious  Disease. — As  a  source  of  direct 
infection,  milk  is  second  only  to  water,  and  deserves  close  hygienic 
attention.  A  large  number  of  infectious  diseases  have  been  traced 
to  milk,  although  the  actual  proof  of  the  etiological  part  played 
by  it  in  such  cases  has  often  been  difficult  to  adduce  and  has  neces- 
sarily been  indirect.  Nevertheless,  even  when  indirect  proof  only 


10  Beijerinck,  Cent,  f.  Bakt,,  vi,  1889. 

"Ward,  Bull.  1«5,  Cornell   Univ.  Agri,  Exp.  Stat.,  1899, 

12  Conn,  Cent.  f.  Bakt,,  ix,  1891. 

"  Wagmann,  Milchztg.,  1890. 


1032  BACTERIA  IN  AIR,   SOIL,   WATER,   AND   MILK 

has  been  brought,  it  has  been  sufficiently  convincing  to  necessitate 
the  most  careful  investigation  into  milk  supplies  whenever  epidemics 
of  certain  infectious  maladies  occur. 

Typhoid-fever  epidemics  have  been  frequently  traced  to  milk 
infection,  and,  in  this  disease,  milk  is,  next  to  water,  the  most 
frequent  etiological  factor.  Schiider,14  in  an  analysis  of  six  hundred 
and  fifty  typhoid  epidemics,  found  four  hundred  and  sixty-two 
attributed  to  water,  one  hundred  and  ten  to  milk,  and  seventy-eight 
to  all  other  causes. 

Trask15  compiled  statistics  of  one  hundred  and  seventy-nine 
typhoid  epidemics  supposed  to  have  been  caused  by  milk,  in  various 
parts  of  the  world.  In  all  such  epidemics  the  origin  of  infection  was 
generally  traceable  to  diseased  or  convalescent  persons  employed  in 
dairies,  to  contaminated  well  water  used  in  washing  milk  utensils, 
or  to  the  use  of  cans  and  bottles  returned  from  dwellings  where 
typhoid  fever  had  existed.  Actual  bacteriological  proof  of  the  in- 
fectiousness  of  milk  by  the  isolation  of  Bacillus  typhosus  is  rare, 
but  has  been  accomplished  in  isolated  instances.  In  the  case  of 
one  epidemic,  Conradi16  isolated  the  bacillus  from  the  milk  on  sale 
at  a  bakery  at  which  a  large  number  of  the  infected  individuals  had 
purchased  their  milk.  The  examination  of  market  milk  at  Chicago, 
through  a  period  of  eight  years,  revealed  the  presence  of  typhoid 
bacilli  but  three  times. 

In  spite  of  the  few  cases  in  which  actual  bacteriological  proof 
has  been  brought,  it  is  not  unlikely  that  careful  and  systematic 
researches  would  reveal  a  far  greater  number,  since  many  writers 
have  shown  that  typhoid  bacilli  may  remain  alive  in  raw  milk  for 
as  long  as  thirty  days,17  and  may  actively  proliferate  in  the  milk 
during  this  time.  One  peculiarity  of  epidemics  which  may  aid  in 
arousing  the  suspicion  that  they  have  originated  in  milk  is  that, 
in  such  cases,  women  and  children  are  far  more  frequently  attacked 
than  men.18 

A  feature  which  adds  considerably  to  the  dangers  of  milk  infec- 
tion is  the  unfortunate  absence  of  any  gross  changes,  such  as  coagula- 
tion, by  the  growth  of  typhoid  bacilli. 

14  Schuder,  Zeit.  f.  Hyg.,  xxxviii,   1901. 

™  Trask,  Bull.  No.  41,  TI.  S.  Pub.  Health  and  Mar.  Hosp.  Serv.,  Wash. 

16  Conradi,  Cent,  f .  Bakt.,  I,  xl,  1905 

17  Heim,  Arb.  a.  d.  kais.  Gesundheitsamt,  v. 

18  Wilckens,  Zeit.  f.  Hyg.,  xxvii,  1898. 


BACTERIA  IN   MILK  1033 

Scarlet  fever,19  though  as  yet  of  unknown  etiology,  has  in  many 
cases  been  traced  indirectly  to  milk  infection.  Trask  has  collected 
fifty-one  epidemics  of  scarlet  fever  presumably  due  to  milk.  In  one 
epidemic  occurring  in  Nor  walk,  Conn.,20  twenty-nine  cases  wer"e 
distributed  among  twenty-five  families  living  in  twenty-four  different 
houses.  The  individuals  affected  did  not  attend  the  same  school, 
and  were  of  entirely  different  social  standing,  the  only  factor  com- 
mon to.  all  of  them  being  the  milk  supply. 

Diphtheria  has  been  frequently  traced  to  the  use  of  infected  milk. 
In  most  of  the  epidemics  reported  as  originating  in  this  way,  the 
proof  has  been  necessarily  indirect.  In  two  out  of  twenty-three 
epidemics  reported  by  Trask,  however,  Bacillus  diphtherias  was 
isolated  from  the  milk  directly.  The  ability  of  the  Klebs-Loeffler 
bacillus  to  proliferate  and  remain  alive  for  a  long  while  in  raw 
milk  has  been  demonstrated  by  Eyre21  and  others. 

Whether  or  not  cholera  asiatica  may  be  transmitted  by  means  of 
milk  has  been  a  disputed  question.  Hesse22  claims  that  cholera 
spirilla  die  out  in  raw  milk  within  twelve  hours.  This  statement, 
however,  has  not  been  borne  out  by  other  observers.23  Unquestion- 
able cases  of  direct  transmission  of  cholera  by  means  of  milk  have 
been  reported  by  a  number  of  writers,  notably  by  Simpson.24 

The  relation  of  milk  to  the  diarrheal  diseases  of  infants  has,  of 
late  years,  received  a  great  deal  of  attention.  In  large  cities,  during 
the  summer  months,  numerous  cases  of  infantile  diarrhea  among 
bottle-fed  babies  occur,  which,  in  many  instances,  are  attributed  to 
feeding  with  contaminated  milk.  Park  and  Holt,25  who  have  made 
extensive  researches  upon  this  question  in  New  York  City,  have 
come  to  the  conclusion  that  the  harmful  effects  of  contaminated  milk 
upon  babies  can  not  be  ascribed  to  any  given  single  microorganism 
in  the  milk.  Specifically  toxic  properties  were  found  by  these  writers 
for  none  of  the  one  hundred  and  thirty-nine  different  species  of 
bacteria  isolated  from  unsterilized  milk.  It  is  unlikely,  therefore, 
that  the  diarrheal  diseases  among  babies  have  a  uniform  bacteriolog- 


18  Trask,  loc.  cit. 

20  Herbert  E.  Smith,  Eep.  Conn.  State  Bd.  of  Health,  1897. 

21  Eyre,  Brit.  Med.  Jour.,  1899. 

22  Hesse,  Zeit.  f.  Hyg.,  xvii,  1894. 
2*Basenau,  Arch.  f.  Hyg.,  xxiii,  1895. 

24  Simpson,  Indian  Med.  Gaz.,  1887. 

25  Park  and  Holt,  Arch,  of  Fed.,  Dec.,  1903. 


1034  BACTERIA  IN   AIR,   SOIL,   WATER,   AND   MILK 

ical  cause.  Whether  or  not  these  diarrheal  conditions  depend  en- 
tirely upon  the  bacterial  contents  of  milk  or,  in  a  large  number 
of  cases  at  least,  upon  the  inability  of  the  child  to  digest  cow 's  milk 
because  of  chemical  conditions,  must  be  left  undecided.  Park  and 
Holt,  in  analyzing  their  extensive  data,  conclude  that  milk  con- 
taining "over  one  million  bacteria  to  the  cubic  centimeter  is  cer- 
tainly harmful  to  the  average  infant." 

The  significance  of  the  presence  of  streptococci  in  milk,  as  an 
element  of  danger,  has  recently  received  much  attention  in  the 
literature.  Heinemann,26  who  has  made  a  careful  comparison  of 
Streptococcus  lacticus  (formerly  spoken  of  as  Bacillus  acidi  lactici 
[Krusel]),  with  other  streptococci,  has  shown  that,  essentially,  this 
streptococcus  does  not  differ  from  streptococci  from  other  sources, 
and  is  practically  indistinguishable  by  cultural  methods  from  Strep- 
tococcus pyo genes.  Similar  comparisons  made  by  Schottmuller,27 
Miiller,28  and  others  have  led  to  like  results.  Since  streptococci  may 
be  found  in  milk  from  perfectly  normal  cows  and  are  almost  regularly 
associated  with  lactic-acid  fermentation,  it  is  unlikely  that  these 
microorganisms  hold  ordinarily  any  specific  relationship  to  disease. 

Recently,  however,  a  number  of  epidemics  of  sore  throat  caused 
by  streptococci  have  been  traced  to  milk  upon  reasonably  reliable 
evidence.  Accounts  of  such  epidemics  in  Chicago  and  in  Baltimore 
have  been  published  by  Capps  and  Miller29  and  by  Hamburger.30 

The  presence  of  pus  cells  and  leucocytes  in  milk,  together  with 
streptococci,  was  also  formerly  regarded  as  of  great  importance. 
Enumerations  of  leucocytes  in  milk  were  first  made  by  Stokes  and 
Weggefarth.31  Their  method  of  enumeration  consisted  in  centri- 
fugalizing  a  definite  volume  of  milk,  spreading  the  entire  sediment 
over  a  definite  area  on  a  slide,  and  counting  the  leucocytes  found 
in  a  number  of  fields.  Calculations  from  this  may  then  be  made 
as  to  the  number  of  leucocytes  per  cubic  centimeter.  This  method, 
and  modifications  of  it,  have  been  used  by  a  large  number  of 
observers,  but  the  value  of  the  conclusions  drawn  from  them  has 
been  much  exaggerated.  Normal  milk  may  contain  leucocytes  in 

26  Heinemann,  Jour.  Inf.  Dis.,  3,  1906. 
"Schottmuller,  Munch,  med.  Woch.,  1903. 

28  Miiller,  Arch,  f .  Hyg.,  Ivi,  1906. 

29  Capps  and  Miller,  Jour.  A.  M.  A.,  June,  1912,  p.  1848. 

30  Hamburger,  Bull,  of  the  Johns  Hopk.  Hosp.,  xxiv,  Jan.,  1913. 
n  Stokes  and  Weggefarth,  Med,  News,  91,  1897. 


BACTERIA  IN   MILK  1035 

moderate  numbers,  and  importance  may  be  attached  to  such  leu- 
cocyte counts  only  when  their  number  largely  exceeds  that  present 
in  other  specimens  of  perfectly  normal  milk.  Whenever  such  high 
leucocyte  counts  are  found,  of  course,  a  careful  veterinary  inspection 
and  examination  for  pyogenic  disease  should  be  made. 

Foot-and-mouth  disease,  an  infectious  condition  prevailing  among 
cattle,  characterized  by  a  vesicular  rash  on  the  mouth  and  about  the 
hoofs,  has,  in  a  number  of  cases,  been  definitely  shown  to  be  trans- 
mitted to  man  through  the  agency  of  milk.  Notter  and  Firth32 
reported  an  epidemic  occurring  among  persons  supplied  with  milk 
from  a  single  dairy  in  which  foot-and-mouth  disease  prevailed  among 
the  cows.  In  this  epidemic,  two  hundred  and  five  individuals  were 
affected  with  vesicular  eruptions  of  the  throat,  with  tonsillitis  and 
swellings  of  the  cervical  lymph  nodes.  Similar  cases  have  been 
reported  by  Pott.33 

Although  anthrax  has  never  been  definitely  shown  to  have  been 
conveyed  by  milk,  Boschetti34  succeeded  in  isolating  living  anthrax 
bacilli  from  a  sample  of  milk  two  weeks  after  its  withdrawal  from 
the  cow. 

Milk  and  Tuberculosis. — The  question  of  the  conveyance  of  tuber- 
culosis by  means  oi  milk  is  a  subject  which,  because  of  its  great 
importance,  has  been  extensively  investigated  by  bacteriologists.  A 
large  number  of  observers  have  succeeded  in  proving  the  presence 
of  tubercle  bacilli  in  tho  milk  of  tuberculous  cows  by  intraperitoneal 
inoculation  of  rabbits  and  guinea-pigs  with  samples  of  milk.  Such 
positive  results  have  been  obtained  by  Bang,35  Hirschberger,36 
Ernst,37  and  many  others.  A  number  of  these  observers,  notably 
Ernst,  have  shown  that  tubercle  bacilli  may  be  present  in  the  milk 
without  tuberculous  disease  of  the  udders.  In  an  examination  of 
the  milk  supply  of  Washington,  D.  C.,38  6.72  per  cent  of  the  samples 
contained  tubercle  bacilli. 

The  path  of  entrance  of  the  bacilli  from  the  cow  into  the  milk 


32  Notter    and    Firth,    quoted    from    Harrington,    ' '  Theory    and    Practice    of 
Hygiene. ' ' 

33  Pott,  Munch,  med.  Woch.,  1899. 
34Hox<>lictti,  fliorn.  mod.  vet.,   1891. 

M  #«./»//,   Dent.  /fit.  f.  Tierchem.,  xi,  1884. 
"Hirwlibrruc);  Deut.  Arch.  f.  klin.  Med.,  xliv,  1889. 

37  Ernst,  H.  C.,  Amer.  Jour.  Med.   Sei.,  xcviii,   1890. 

38  Anderson,  Bull.  No.  41,  U.  S.  Pub.  Health  and  Mar.  Hosp.  Serv.,  Wash.,  1908. 


1036  BACTERIA  IN  AIR,   SOIL,   WATER,   AND   MILK 

has  long  been  a  subject  of  controversy.  That  the  bacilli  may  easily 
enter  the  milk,  when  tuberculous  disease  o'f  the  udder  is  present, 
stands  to  reason  and  is  universally  conceded.  It  is  now  believed 
also,  on  the  basis  of  much  experimentation,  that  in  systemically 
infected  cows  tubercle  bacilli  may  pass  through  the  mammary  glands 
into  the  milk,  without  evidence  of  local  disease  in  the  secreting 
gland.  An  experiment  performed  by  the  Royal  British  Tuberculosis 
Commission39  illustrates  this  point.  A  cow,  injected  subcutancously 
with  tubercle  bacilli  behind  the  shoulder,  began  to  discharge  tubercle 
bacilli  in  the  milk  within  seven  days  after  inoculation  and  continued 
to  do  so  until  death  from  generalized  tuberculosis. 

Milk  may  become  indirectly  contaminated,  furthermore,  with 
tubercle  bacilli  emanating  from  the  feces  of  cows.  It  has  been 
shown  that  tubercle  bacilli  are  present  in  the  feces  of  cattle  so  early 
in  the  disease  that  diagnosis  can  be  made  only  by  a  tuberculin 
test.40 

Whether  or  not  contaminated  milk  is  common  as  an  etiological 
factor  in  human  tuberculosis,  must  be  considered  at  present  as  an 
unsettled  question.  Behring,  at  the  Congress  of  Veterinary  Medicine, 
at  Cassel,  in  1903,  advanced  the  view  that  pulmonary  tuberculosis 
in  adults  may  be  a  late  manifestation  of  a  milk  infection  contracted 
during  infancy.  He  stated  as  his  own  opinion,  moreover,  that  most 
cases  of  tuberculosis  in  man  are  traceable  to  this  origin.  The  problem 
is  as  difficult  of  solution  as  it  is  important.  In  bottle-fed  infants, 
infection  by  means  of  milk  unquestionably  occurs  with  considerable 
frequency.  Smith,41  Kossel,  Weber,  and  Huess,42  and  others,  have 
isolated  tubercle  bacilli  of  the  bovine  type  from  the  mesenteric 
lymph  nodes  of  many  infected  children.  Animal  experimentation 
has,  furthermore,  revealed  that  lesions  in  the  mesenteric  nodes, 
as  well  as  later  in  the  bronchial  lymph  nodes,  may  occur  as  a 
consequence  of  feeding  tubercle  bacilli,  without  any  demonstrable 
lesions  in  the  intestinal  mucosa.  It  is  thus  certain  that  infection 
by  the  ingestion  of  tuberculous  milk  may  occur,  especially  among 
young  children  who,  as  is  well-known,  are  comparatively  susceptible 
to  bacilli  of  the  bovine  type.  Whether  or  not  such  infection  will 


30  Quoted  from  Mohlcr,  P.  TT.,  and  Mar.  Hosp.  Serv.  Bull.  41,  1908. 

40  KcJirocdcr  and  Cotton,  Bull.  Bureau  Animal  Industry,  Wash.,  1007. 

41  8 milk,  Trans.  Assn.  Ainer.  Physic.,  18,  ]JM):'.. 

4- Kossel,  Weber,  and  Huess,  Tubcrkul.  Arb.  a.  d.  kais.  Gesimdheitsamt,  1904, 
1905,  Hft.  1  and  3. 


BACTERIA  IN   MILK  1037' 

account  for  many  cases  of  tuberculosis  in  adults  is  a  question  which, 
for  final  solution,  will  require  much  more  investigation.  The  sole 
reliable  method  of  approaching  it  lies  in  determining  the  type, 
human  or  bovine,  of  the  bacilli  present  in  a  large  number  of  cases. 
Experience  thus  far  seems  to  indicate  that  the  bovine  type  is  com- 
paratively rare  in  the  pulmonary  disease  of  adults. 

The  value  of  the  tuberculin  reaction  for  diagnosis,  and  the 
elimination  of  all  cattle  showing  a  positive  reaction,  for  the  preven- 
tion of  tuberculosis,  can  not  be  overestimated.  The  failure  of  the 
test  in  diseased  animals  is  rare,  and  an  accurate  diagnosis  can  be 
established  in  over  90  per  cent  of  diseased  animals.43  The  assertion 
that  the  cattle  are  permanently  injured  by  tuberculin  injections  is 
without  scientific  basis.  If  this  test  were  conscientiously  carried 
out,  and  infected  cattle  condemned,  the  dangers  from  bovine  bacillus 
infection  would  be  practically  eliminated,  for  there  are  but  few 
instances  in  which  science  has  been  able  to  furnish  such  definite 
information  for  absolute  protection.  It  is  needless  to  say,  however, 
that  the  carrying  out  of  such  precautions  is  subject  to  great  expense 
and  great  difficulties  of  organization. 

Dairy  inspection  is  practiced  in  the  vicinity  of  many  of  our  larger 
cities,  and  the  movement  is  daily  gaining  ground.  Until  fully  estab- 
lished, however,  upon  a  financial  basis  which  brings  the  best  products 
within  the  means  of  the  poorer  classes,  other  inexpensive  measures 
to  render  milk  safe  must  often  be  resorted  to. 

Sterilization  by  high  temperatures  is  objected  to  by  pediatricians 
because  of  the  physical  and  chemical  changes  produced  in  the  milk 
which  are  said  to  detract  from  its  nutritive  value. 

The  development  of  scurvy  and  rickets  in  infants  has  often  been 
attributed  to  the  use  of  such  milk.  These  objections,  however,  do 
not  apply  to  the  use  of  milk  which  has  been  subjected  to  the  process 
of  ' 'pasteurization."  By  this  term  is  meant  the  heating  of  any 
substance  to  60°  C.  for  twenty  to  thirty  minutes.  The  process,  first 
devised  by  Pasteur  for  the  purpose  of  destroying  germs  in  wine 
and  beer  in  which  excessive  heating  was  supposed  to  injure  flavor, 
brings  about  the  death  of  all  microorganisms  which  do  not  form 
spores — in  other  words,  of  all  the  bacteria  likely  to  be  found  in 
milk  which  can  give  rise  to  infection  per  os.  At  the  same  time  the 
chemical  and  physical  constitution  of  the  milk  is  not  appreciably 

"Hohler,  loc.  cit. 


1038  BACTERIA   IN   AIR,   SOIL,   WATER,   AND   MILK 

changed,  at  least  not  to  an  extent  which  renders  it  less  valuable 
as  a  food.  Statistics  by  Park  and  Holt  have  shown  strikingly  the 
advantages  of  pasteurized  over  raw  milk  in  infant  feeding.  Of 
fifty-one  children  fed  with  raw  milk  during  the  summer  months, 
thirty-three  had  diarrhea,  two  died,  and  only  seventeen  remained 
entirely  well.  Of  forty-one  receiving  pasteurized  milk,  but  ten  had 
diarrhea,  one  died,  and  thirty-one  remained  entirely  well  throughout 
the  summer.  The  actual  diminution  of  the  living  bacterial  contents 
of  milk  by  pasteurization  is  enormous,  the  milk  so  treated  often 
containing  not  more  than  one  thousand,  usually  less  than  fifteen 
thousand,  living  bacteria  to  each  cubic  centimeter. 

Methods  of  Estimating  the  Number  of  Bacteria  in  Milk. — In  es- 
timating the  number  of  bacteria  in  milk,  colony  counting  in  agar 
or  gelatin  plates  is  resorted  to.  Great  care  must  be  exercised  in 
obtaining  the  specimens.  If  taken  from  a  can,  the  contents  of  the 
can  should  be  thoroughly  mixed,  since  the  cream  usually  contains 
many  more  bacteria  than  the  rest  of  the  milk.  The  specimen  is  then 
taken  into  a  sterile  test  tube  or  flask.  If  the  milk  is  supplied  in  an 
ordinary  milk  bottle,  this  should  be  thoroughly  shaken  before  being 
opened,  and  the  specimen  for  examination  taken  out  with  a  sterile 
pipette.  Dilutions  of  the  specimen  can  then  be  made  in  sterile  broth 
or  salt  solution.  If  an  initial  dilution  of  1 :100  is  made,  quantities 
ranging  from  1  c.c.  to  0.1  c.c.  of  this  will  furnish  0.01  c.c.  to  0.001  c.c. 
of  the  milk,  respectively.  Inoculation  of  properly  cooled  tubes  of 
melted  neutral  agar  and  gelatin,  with  varying  quantities  of  these 
dilutions,  are  then  made  and  plates  poured.  After  twenty-four  to 
forty-eight  hours  at  room  temperature  or  in  the  incubator,  colony 
counting  is  done,  and  the  proper  calculation  is  made.  In  samples 
in  which  few  bacteria  are  expected,  direct  transference  of  1/20  or 
1/40  of  a  c.c.  of  the  whole  milk  into  the  agar  may  be  made.  This 
method  saves  time  but  is  less  accurate. 

Direct  Methods  of  Counting  Bacteria. — Direct  methods  of  count- 
ing bacteria  in  milk  have  recently  been  advised,  the  one  most 
extensively  tried  being  that  of  Prescott  and  Breed.  By  this  method 
a  capillary  tube  is  marked  to  measure  accurately  0.01  c.c.  This 
amount  of  the  milk  is  spread  over  a  square  cm.  on  a  microscope  slide. 
It  is  dried  in  the  air  and  fixed  with  methyl  alcohol,  after  which  the 
fatty  constituents  can  be  dissolved  with  xylol.  It  can  then  be  stained 
lightly  with  the  Jenner  stain.  The  bacteria  are  counted  under  an 
oil  immersion  lens,  the  tube  length  and  magnification  being  so 


BACTERIA   IN  MILK  1039 

arranged  that  the  microscopic  field  covers  1/50  sq.  mm.  A  standard- 
ized eyepiece  micrometer  may  be  used.  The  average  number  of 
bacteria  found  in  such  fields  may  be  multiplied  by  5,000  to  give 
the  number  of  bacteria  contained  in  0.01  c.c.  of  milk.  This  method 
nas  not  yet  displaced  the  one  of  plating  and  does  not  promise  to 
do  so  for  some  time. 

For  the  isolation  of  special  pathogenic  bacteria  from  milk,  no 
rules  can  be  laid  down,  since,  in  every  case,  the  method  adapted 
to  the  particular  organism  sought  for  must  be  chosen. 

Tubercle  bacilli  can  be  isolated  from  milk  with  success  only  by 
guinea-pig  injection.  The  milk  is  centrifugalized  and  5  c.c.  of  the 
sediment,  together  with  some  of  the  cream  that  has  risen  to  the  top, 
is  intraperitoneally  or  subcutaneously  injected. 

The  control  of  milk  in  the  market  depends  upon  careful  regula- 
tions, which  must  include  care  of  cattle,  dairy  inspection  and  bac- 
teriological control  of  the  delivered  milk.  This  is  a  subject  which 
is  too  extensive  to  touch  upon  in  a  book  of  this  kind.  However, 
a  general  idea  of  the  methods  employed  may  be  obtained  by  studying 
the  accompanying  table,  which  is  taken  from  the  New  York  City 
Department  of  Health  Regulation  for  the  Sale  of  Milk  and  Cream. 

Bacteria  and  Butter. — Butter  is  made  from  cream  separated  from 
milk  either  by  standing  or  by  centrifugalization.  After  this,  the 
cream  is  agitated  by  churning,  which  brings  the  small  fat-globules 
into  mutual  contact,  allows  them  to  adhere  to  each  other  and  form 
clumps  of  butter.  It  has  been  a  matter  of  common  experience,  how- 
ever, that  unless  the  cream  is  allowed  to  "ripen"  for  a  considerable 
period  before  churning,  the  resulting  butter  lacks  the  particular 
quality  of  flavor  which  gives  it  its  market  value.  The  interval  of 
ripening,  at  first  a  necessity  upon  small  farms  where  cream  must 
be  collected  and  allowed  to  accumulate,  has  now  been  recognized 
as  an  essential  for  the  production  of  the  best  grades  of  butter,  and 
it  has  been  shown  that  the  changes  taking  place  in  the  cream  during 
this  period  are  referable  to  the  action  of  bacteria.  Cream,  which 
before  the  ripening  process  contains  but  50,000  bacteria  to  each  cubic 
centimeter,  at  the  end  of  a  period  of  " ripening"  will  often  contain 
many  millions  of  microorganisms.  At  the  same  time,  the  cream 
becomes  thick  and  often  sour. 

The  species  of  bacteria  which  take  part  in  this  process  and  which, 
therefore,  must  determine  to  a  large  extent  the  quality  of  the  end 
product,  are  various  and,  as  yet,  incompletely  known.  Usually  some 


1040 


BACTERIA  IN  AIR,   SOIL,   WATER,  AND   MILK 


REGULATIONS  GOVERNING  THE  GRADES  AND  DESIGNATION  OF  MILK 
The  following  classifications  apply  to  milk  and  cream.     The  regulations  regarding 


GRADES  OF 
MILK  OR 
CREAM 
WHICH  MAY 
BE  SOLD  IN 
THE  CITY  OF 
NEW  YORK 

DEFINITION 

TUBERCULIN 
TEST  AND 
PHYSICAL 
CONDITION 

BACTERIAL  CONTENTS 

GRADE  A 

Milk  or  Cream 
(Raw) 

Grade  A  milk  or  cream  (raw)  is  milk  or 
cream  produced  and  handled  in  accord- 
ance with  the  minimum  requirements, 
rules  and  regulations  as  herein  set  forth. 

1.  Only    such 
cows  shall  be  ad- 
mitted   to    the 
herd  as  have  not 
reacted  to  a 
diagnostic  injec- 
tion of  tubercu- 
lin   and    are    in 
good  physical 
condition. 
2.  All   cows 
shall    be    tested 
annually    with 
tuberculin     and 
all  reacting  ani- 
mals shall  be  ex- 
cluded from  the 
herd. 

Grade  A  milk  (Raw)  shall  not  con- 
tain more  than  60,000  bacteria  per 
c.c.  and  cream  more  than  300,000 
bacteria  per  c.c.  when  delivered  to 
the  consumer  or  at  any  time  prior  to 
such  delivery. 

Milk  or  Cream 
(Pasteurized) 

Grade  A  milk  or  cream  (pasteurized)  is 
milk  or  cream  handled  and  sold  by  dealers 
holding  permits  therefor  from  the  Board 
of  Health,  and  produced  and  handled  in 
accordance  with  the  requirements,  rules 
and  regulations  as  herein  sot  forth. 

No  tuberculin 
test  required  but 
cows  must  bo 
healthy  as  dis- 
clossd  by  phys- 
ical examination 
made  annually. 

Grade  A  milk   (pasteurized)   shall 
not  contain  more  than  30,000  bacteria 
per  c.c.  and  Cream  (pasteurized)  more 
than  150,000  bacteria  psr  c.c.  when 
delivered  to  the  consumer  or  at  any 
time  after   pasteurization   and   prior 
to  such  delivery. 
No   milk   supply    averaging    more 
than  200,000  bacteria  per  c.c.  shall  be 
pasteurized  for  sale  under  this  desig- 
nation. 

GRADE  B 

Milk  or  Cream 
(Pasteurized) 

Grade  B  milk  or  cream  (pasteurized)  is 
milk  or  cream  produced  and  handled  in 
accordance  with  the  minimum  require- 
ments, rules  and  regulations  herein  set 
forth  and  which  has  been  pasteurized  in 
accordance  with  the  requirements  and 
rules  and  regulations  of  the  Department 
of  Health  for  pasteurization. 

No  tuberculin 
tests  required  but 
cows    must    be 
healthy  as  dis- 
closed by  phys- 
ical examination 
made  annually. 

No  milk  under  this  grade  shall  con- 
tain more  than  100,000  bacteria  per 
c.c.  and  no  cream  shall  contain  more 
than  500,000  bacteria  per  c.c.  when 
delivered  to  the  consumer  or  at  any 
time  after  pasteurization  and  prior  to 
such  delivery. 
No   milk   supply   averaging   more 
than  1,500,000  bacteria  per  c.c.  shall 
be  pasteurized  in  this  city  for  sale 
under  this  designation. 
No   milk   supply   averaging   more 
than  300,000  bacteria  per  c.c.  shall  be 
pasteurized  outside  of  this  city  for 
sale  under  this  designation. 

GRADE  C 
Milk  or  Cream 

(Pasteurized) 
(For  cooking 
and    manu- 
facturing  pur- 
poses only.) 

Grade  C  milk  or  cream  is  milk  or  cream 
not  conforming  to  the  requirements  of 
any  of  the  subdivisions  of  Grade  A  or 
Grade  B  and  which  has  been  pasteurized 
according  to  the  requirements  and  rules 
and  regulations  of  the  Board  of  Health  or 
boiled  for  at  least  two  (2)  minutes. 

No  tuberculin 
test  required  but 
cows  must  be 
healthy  as  dis- 
closed by  phys- 
ical examination 
made  annually. 

No  milk  of  this  grade  shall  contain 
more  than  300,000  bacteria  per  c.c. 
and  no  cream  of  this  grade  shall  con- 
tain more  than  1,500,000  bacteria 
per  c.c.  after  pasteurization. 

NOTE — Sour  milk,  buttermilk,  sour  cream,  kumyss,  matzoon,  zoolac  and  similar  products  shall  not  be  made 
the  process  of  souring.  Sour  cream  shall  not  contain  a  less  percentage  of  fats  than  that  designated  for  cream. 

No  other  words  than  those  designated  herein  shall  appear  on  the  label  of  any  container  containing  milk  or  cream 

The  tsrm  "certified"  milk  is  usually  defined  for  each  region  by  a  special  commission  of  the  County  Med.  Soc., 
County,  N.  Y.: 

CERTIFIED  MILK  must  have  every  characteristic  of  pure,  clean,  fresh,  wholesome  cow's  milk.  The  milk  must 
Nothing  must  be  added  to  the  milk  and  nothing  taken  awav. 

CERTIFIED  MILK  shall  not  contain  less- than  4  ner  cent  of  butter  fat. 

*  Table  taken  from  Rules  and  Regulations  of  N.  Y.  City  Department  of  Health,  1914,  applying  to  sale  of  milk 


BACTERIA   IN   MILK 


1041 


AND  CREAM  WHICH  MAY  BE  SOLD  IN  THE  CITY  OF  NEW  YORK  * 

bacterial  content  and  time  of  delivery  shall  not  apply  to  sour  cream 


NECES- 

SARY 

SCORES 
FOR 
DAIRIES 

TIME  OF 
DELIVERY 

BOTTLING 

LABELING 

PASTEURIZA- 
TION 

PRODUC- 

ING 

Unless    other- 

wise   specified    in 

Equip.  25 

Shall    be    de- 
livered  within 

the     permit     this 
milk    or   cream 

Outer  caps  of  bottles  shall  be  white  and 
shall  contain  the  words  Grade  A,  Raw, 

Meth.  50 

36    hours    after 

shall  be  delivered 

in  black  letters  in  large  type,  and  shall 

production. 

to    the    consumer 

state  the  name  and  address  of  the  dealer. 

Total  75 

only  in  bottles. 

Equip.  25 
Meth.  43 
Total  68 

Shall    be    de- 
livered     within 
36    hours    after 
pasteurization. 

Unless   other- 
wise   specified    in 
the     permit     this 
milk      or      cream 
shall   be  delivered 
to    the    consumer 
only  in  bottles. 

Outer  caps  of  bottles  shall  be  white  and 
shall  contain  the  words  Grade  A  in  black 
letters  in  large  type,  date  and  hours  be- 
tween   which    pasteurization    was  com- 
pleted;   place  where  pasteurization  was 
performed;  name  of  the  person,  firm  or  the 
corporation  offering  for  sale,  selling  or  de- 

Only such 
milk    or     cream 
shall  be  regarded 
as  pasteurized  as 
has     been     sub- 
jected to  a  tem- 
perature  averag- 

livering same. 

ing  145°  Fahr.  for 

not  less  than  30 

minutes. 

Outer  caps  of  bottles  containing  milk 
and  tags  affixed  to  cans  containing  milk 

or   cream   shall   be   white   and   marked 

Only      such 

"Grade  B"   in   bright   green   letters  in 

milk     or     cream 

Equip.  20 

Milk   shall  be 
delivered  within 

large  type,  date  pasteurization  was  com- 
pleted,  place   where   pasteurization   was 

shall  be  regarded 
as  pasteurized  as 

Meth.  35 

36     hours     and 

May    be    deliv- 

performed, name  of  the  person,  firm  or 

has     been     sub- 

cream within  48 

ered    in    cans    or 

corporation  offering  for  sale,  selling  or  de- 

jected to  a  tem- 

Total 55 

hours  after  pas- 
teurization. 

bottles. 

livering  same.    Bottles  containing  creams 
shall  be  labeled  with  caps  marked  "Grade 

perature    averag- 
ing 145°  Fahr.  for 

B"  in  bright  green  letters,  in  large  type 
and  shall  give  the  place  and  date  of  bot- 

not less  than  30 
minutes. 

tling  and  shall  give  the  name  of  person, 

firm  or  corporation  offering  for  sale,  selling 

or  delivering  same. 

Only    such 

milk    or     cream 

Shall    be    de- 

Tags affixed  to  cans  shall  be  white  and 

shall  be  regarded 

livered       within 

May    be    deliv- 

shall be  marked  in  red  with  the  words 

as  pasteurized  as 

Score  40 

48    hours    after 

ered  in  cans  only. 

"Grade  C"  in  large  type  and  "for  cook- 

has    been     sub- 

pasteurization. 

ing"  in  plainly  visible  type,  and  cans  shall 
have  properly  sealed  metal  collars,  painted 

jected  to  a  tem- 
perature  averag- 

red on  necks. 

ing  145°  Fahr.  for 

not  less  than  30 

minutes. 

from  any  milk  of  a  less  grade  than  that  designated  for  "Grade  B"  and  shall  be  pasteurized  before  being  put  through 

or  milk  or  cream  products  except  the  word  "certified"  when  authorized  under  the  State  laws. 

sanction  by  State  Law.      The  following  is  the  definition  of  certified  milk  given  by  the  Milk  Commission  of  Kings 

be  in  its  natural  state,  not  having  been  heated  and  without  the  addition  of  coloring  matter  or  preservatives, 
and  cream. 


1042  BACTERIA   IN   AIR,   SOIL,   WATER,   AND   MILK 

variety  of  lactic-acid  bacilli  is  present  and  these,  as  in  milk,  outgrow 
other  species  and,  according  to  Conn,44  are  probably  essential  for 
' '  ripening. ' ' 

It  would  be  of  great  practical  value,  therefore,  if  definite  pure 
cultures  of  the  bacteria  which  favor  the  production  of  agreeable 
flavors  could  be  distributed  among  dairies.  In  Denmark  this  has 
been  attempted  by  first  pasteurizing  the  cream  and  then  adding 
a  culture  of  bacteria  isolated  from  "  favorable "  cream.  These  cul- 
tures, delivered  to  the  dairyman,  are  planted  in  sterilized  milk,  in 
order  to  increase  their  quantity,  and  this  culture  is  then  poured 
into  the  pasteurized  cream.  In  most  cases,  these  so-called  " starters" 
are  not  pure  cultures,  but  mixtures  of  three  or  more  species  derived 
from  the  original  cream. 

Adverse  accidents  in  the  course  of  butter-making,  such  as  "sour- 
ing" or  "bittering"  of  butter,  are  due  to  the  presence  of  con- 
taminating, probably  proteolytic,  microorganisms  in  the  cream 
during  the  process  of  "ripening." 

As  a  means  of  transmitting  infectious  disease,  butter  is  of  im- 
portance only  in  relation  to  tuberculosis.  Obermuller,45  Rabino- 
witch,46  Boyce,47  and  others,  have  repeatedly  found  tubercle  bacilli 
in  market  butter,  and  Mohler,48  Washburn,  and  Rogers  have  recently 
shown  that  these  bacilli  could  remain  alive  and  virulent  for  as  long 
as  five  months  in  butter  kept  at  refrigerator  temperature.  The 
acid-fast  butter  bacillus,  described  by  Rabinowitch  as  similar  to 
the  true  Bacillus  tuberculosis,  shows  decided  cultural  and  mor- 
phological differences  from  the  latter. 

Bacteria  and  Cheese. — The  conversion  of  milk  products  into 
cheese  consists  in  a  process  of  protein  decomposition  which,  by  its 
end  products,  leucin,  tyrosin,  and  ammonia  compounds,  largely 
determines  the  cheese-flavors.  The  production  of  cheese,  therefore, 
is  due  to  the  action  of  proteolytic  bacterial  enzymes49  and  the  variety 
of  a  cheese  is  largely  determined  by  the  microorganisms  which  are 
present  and  by  the  cultural  conditions  prevailing.  The  sterilization 

"Conn,  "Agricultural  Bacteriology,"  Phila.,  1901. 
45  Obermuller,  Hyg.  Eundschau,   14,   1897. 
*•  Rabinowitch,  Zeit.  f.  Hyg.,  xxvi,  1897. 

47  Boyce  and  Woodhead,  Brit.  Med.  Jour.,  2,  1897. 

48  Mohler,  U.  S.  P.  H.  and  Mar.  Hosp.  Serv.  Bull.  41,  1908. 

49  Freudenreich,  Koch's  Jahresbericht,  etc.,  135,  1891. 


BACTERIA  IN   MILK  1043 

of  cream,  or  the  addition  of  antiseptics,  absolutely  prevents  cheese 
production. 

The  organisms  which  arc  concerned  in  such  processes  have  been 
extensively  studied  and  attempts  have  been  made,  with  moderate 
success,  to  produce  a  definite  flavor  with  pure  cultures. 

In  the  production  of  cheese  the  two  varieties,  hard  and  soft 
cheeses,  depend  not  so  much  upon  the  bacterial  varieties  as  upon 
the  differences  in  the  treatment  of  the  curds  before  bacterial  action 
has  begun.  In  the  former  case,  a  complete  freeing  of  the  curds  from, 
the  whey  furnishes  a  culture  medium  which  is  comparatively  dry 
and  of  almost  exclusively  protein  composition;  in  the  latter,  reten- 
tion of  whey  gives  rise  to  cultural  conditions  in  which  more  rapid 
and  complete  bacterial  action  may  take  place.  The  holes,  which 
are  so  often  observed  in  some  of  the  hard  cheeses,  are  due  to  gas 
production  during  the  process  of  "ripening." 

As  to  the  varieties  of  microorganisms  present  in  various  cheeses, 
much  careful  work  has  been  done.  Duclaux50  attributed  the  "ripen- 
ing" of  some  of  the  soft  cheeses  to  a  microorganism  closely  related 
to  Bacillus  subtilis.  V.  Freudenreich51  in  part  substantiated  this, 
but  laid  particular  stress  upon  the  action  of  Oidium  lactis,  a  mold, 
and  upon  several  varieties  of  yeast.  Conn,52  more  recently,  in  a 
bacteriological  study  of  Camembert  cheese,  has  demonstrated  that 
the  production  of  this  cheese  depends  upon  the  united  action  of  two 
microorganisms,  one  an  oi'dium,  like  the  Oidium  lactis  of  Freuden- 
reich, which  is  found  chiefly  in  the  interior  softened  areas,  the  other 
a  mold  belonging  to  the  penicillium  variety,  found  in  a  matted  felt- 
work  over  the  surface  and  penetrating  but  a  short  distance.  In 
spite  of  the  scientific  basis  upon  which  the  work  of  these  men  and 
of  others  has  seemed  to  place  cheese  production,  attempts  at  uni- 
formity in  cheese  production  have  met  with  almost  insuperable 
obstacles  because  of  the  presence  of  a  variety  of  adventitious  micro- 
organisms which,  depending  in  species  and  proportion  upon  the  local 
conditions  under  which  the  various  cheeses  have  been  produced, 
have  added  minor  characteristics  of  flavor  which  have  determined 
market  value.  Occasional  failure  of  good  results  in  cheese  produc- 


60 Duclaux.  "Le  Lait,"  Paris,  1887. 

61  V.  Freudenreich,  Cent,  f .  Bakt.,  II,  i,  1895. 

*-C&nn,  Bull.  Statis.  Agri.  Exp.  Stat.  35,  1905. 


1044  BACTERIA   IN  AIR,   SOIL,   WATER,   AND   MILK 

tion53  is  due  to  contamination  with  other  chromogenic  or  putrefactive 
bacteria. 

In  its  relationship  to  the  spread  of  infectious  disease,  cheese  is 
relatively  unimportant  except  in  regard  to  tuberculosis.  Typhoid 
and  other  non-spore  forming  pathogenic  germs  can  not  survive  the 
conditions  existing  during  cheese-ripening  for  any  length  of  time. 
Tubercle  bacilli,  both  of  the  human  and  bovine  types,  have  been 
found  in  cheese  by  Harrison54  and  others,  and  Galtier  has  shown 
experimentally  that  tubercle  bacilli  may  remain  alive  and  virulent 
in  both  salted  and  unsalted  cheese  for  as  long  as  ten  days. 


THE   LACTIC-ACID   BACILLI  AND   METCHNIKOFF'S* 
BACTERIOTHERAPY 

A  problem  which  has  occupied  clinical  investigation  for  many 
years  is  that  of  gastrointestinal  autointoxication.  There  are  a 
number  of  conditions  occurring  in  man,  in  which  symptoms  pro- 
foundly affecting  the  nervous  system,  the  circulation,  and,  in  a 
variety  of  ways,  the  entire  body,  can  be  clinically  traced  to  the 
intestines,  and  can,  in  many  cases,  be  relieved  by  thorough  purgation 
and  careful  diet.  In  some  of  these  conditions,  specific  microorgan- 
isms can  be  held  accountable  for  the  diseases  (B.  enteritidis,  B. 
botulinus,  etc.).  In  other  cases,  however,  etiological  investigations 
have  met  with  but  partial  success  because  of  the  large  variety  of 
microorganisms  present  in  the  intestinal  tract  and  because  of  the 
complicated  symbiotic  conditions  thereby  produced.  Intestinal 
putrefaction,  recognized  as  the  cardinal  feature  of  such  maladies, 
has  been  attributed  to  Bacillus  proteus  vulgaris,55  to  Bacillus 
aerogenes  capsulatus,  to  Bacillus  putrificus,56  and  to  a  number  of 
other  bacteria,  but  definite  and  satisfactory  proof  as  to  the 
etiological  importance  of  any  of  these  germs  has  not  yet  been 
advanced.  The  fact  remains,  however,  that,  whatever  may  be  the 

*  See  also  B.  Acidophilus,  etc.,  in  another  section  of  this  book. 
M Beijerinck,  Koch's  Jahresber,  etc.,  82,  189. 

"Harrison  and  Galtier,  quoted  from  Mohler,  U.  S.  Pub.  H.  and  Mar.  Hosp. 
Serv.,  Hygiene  Lab.  Bull.  41,  1908. 
65  Lesage,  Eev.  de  med.,  1887. 
64  Tissier,  Ann.  de  1  'inst.  Pasteur,  1-905. 


BACTERIA  IN   MILK  1046 

specific  cause,  the  disease  itself,  a  grave  and  often  fatal  affliction, 
may  be  clinically  traced,  in  a  number  of  cases,  to  the  absorption 
of  poisons  from  the  intestinal  canal,  and  it  is  more  than  likely  that 
these  poisons  are  the  products  of  bacterial  activity.  Reason  dictates, 
furthermore,  that  the  bacteria  primarily  responsible  for  the  produc- 
tion of  these  toxic  substances  do  not  belong  to  the  varieties  which 
attack  carbohydrates  only,  but  must  belong  to  that  class  of  aerobic 
and  anaerobic  germs  which  possess  the  power  of  breaking  up  proteins 
—in  other  words,  the  bacteria  of  putrefaction. 

On  the  basis  of  the  mutual  antagonism  existing  in  culture  be- 
tween many  acid-producing  bacteria  and  those  of  putrefaction — a 
phenomenon  recognized  by  some  of  the  earliest  workers  in  this 
field,  many  investigators  have  suggested  the  possibility  of  combating 
intestinal  putrefaction  by  adding  acid-forming  bacteria  together 
with  carbohydrates  to  the  diet  of  patients  suffering  from  this  condi- 
tion. The  first  to  suggest  this  therapy  was  Escherich57  who  proposed 
the  use,  in  this  way,  of  Bacillus  lactis  aerogenes;  with  the  same 
end  in  view,  Quincke,58  a  little  later,  suggested  the  use  of  yeasts — 
Oi'dium  lactis.  The  reasoning  underlying  these  attempts  was  mean- 
while upheld  by  experiments  carried  out  both  in  vitro  and  upon  the 
living  patient.  Thus  Brudzinski5^  was  able  to  demonstrate  that 
Bacillus  lactis  aerogenes,  in  culture,  inhibited  the  development  of 
certain  races  of  the  proteus  species  and  succeeded  in  obtaining 
markedly  favorable  results  by  feeding  pure  cultures  of  Bacillus 
lactis  aerogenes  to  infants  suffering  from  fetid  diarrhea.  Similar 
experiments60  carried  out  with  the  Welch  bacillus  (aerogenes  cap- 
sulatus)  and  Bacillus  coli,  however,  had  no  such  corroboratory 
results,  since  this  anaerobe  possesses  a  considerable  resistance  against 
an  acid  reaction.  In  considering  the  difficulties  of  the  problems  in- 
volved in  this  question,  it  occurred  to  Metchnikoff61  that  much  of 
the  practical  failure  of  therapy,  based  upon  the  principles  stated 
above,  might  be  referred  to  insufficent  powers  of  acid  production 
on  the  part  of  Bacillus  coli,  Bacillus  lactis  aerogenes,  and  other  germs 

57  Escherich,  Therapeut.  Monatshefte,  Oct.,  1887. 

**Quincke,  Verhandl.  des  Congress  f.  Inn.  Med.,  Wiesbaden,  1898. 

69  Brudzinski,  Jahrbuch  f.  Kinderheilkunde,  52,   1900    (Erganzungsheft). 

80  Tissier  and  Martelly,  Ann.  de  1  'inst.  Pasteur,  1906. 

n Metchnikoff,  "Prolongation  of  Life/'  G.  P.  Putnam's  Sons,  N.  Y.;  also  .in 
' '  Bacteriotherapie, "  etc.  "Bibliotheque  de  therapeutique,  "  Gilbert  and  Carnot, 
Paris,  1909. 


1046 


BACTERIA  IN  AIR,   SOIL,   WATER,   AND   MILK 


\ 


previously  use'd.  In  searching  for  more  powerful  acid  producers, 
his  attention  was  attracted  to  Bacillus  bulgaricus,  isolated  from 
milk  by  Massol62  and  Cohendy63  in  1905.  This  bacillus,  according 
to  the  researches  of  Bertrand  and  Weisweiller,6*  produces  as  much 
as  25  grams  of  lactic  acid  per  liter  of  milk.  In  addition  to  this, 

it  manufactures,  from  the  same 
quantity  of  milk,  about  50  cen- 
tigrams of  acetic  and  succinic 
acids  and  exerts  no  putre- 
factive action  upon  proteins. 
Added  to  these  characters,  it 
is  especially  adapted  to  thera- 
peutic application  by  its  com- 
plete lack  of  pathogenicity. 

The  administration  of  the 
bacillus  to  patients  suffering 
from  intestinal  putrefaction, 
first  suggested  by  Metchnikoff 
in  1906,  has,  since  that  time, 
been  extensively  practiced  and 
often  with  remarkable  success. 
In  spite  of  sharp  criticism, 
especially  by  Luersen  and 
Kiihn,65  who  deny  much  of 
the  antiputrefactive  activity 
of  the  bacillus,  the  treatment 
of  Metchnikoff  has  found 
many  adherents,  upon  the 
basis  of  purely  clinical  experi- 
ment. It  is  not  possible  to  review  completely  the  already  extensive 
literature.  Among  the  more  valuable  contributions  may  be  mentioned 
the  articles  by  Grekoff,66  by  Wegele,67  and  by  Klotz.68  In  Metchni- 
koff's  experiments  and  in  the  work  of  his  immediate  successors,  the 


FIG.  119. — BACILLUS  BULGARICUS. 


™Massol,  Eevue  medicale  de  la  Suisse  romande,  1905. 
63  Cohendy,  Comptes  rend,  de  la  soc.  de  biol.,  60,  1906. 


"Bertrand  and   Weisweiller,  Ann.  de  Pinst.  Pasteur,  1906. 
85  Luersen  and  Kuhn,  Cent.  f.  Bakt.,  II,  xx,  1908. 

"Grckoff,  "  Observations  cliniques  sur  1'effet  du  lact.  agri.,"  etc.,  St.  Peters- 
burg,  1907. 

«7  Wcyele,  Deut.  med.  Woch.,  xxxiv,  1908. 
"Klotz,  Zentralbl.  f.  innere  Med.,  1908. 


BACTERIA  IN  ILKM  1047 

bacillus  was  used  cither  in  milk  culture  or  in  broth  in  which  it  was 
induced  to  grow  in  symbiosis  with  other  microorganisms. 

BACTERIOLOGICAL   EXAMINATION  OF    OYSTERS 

On  account  of  the  danger  of  the  transmission  of  typhoid  fever 
by  oysters  which  have  been  bred  or  stored  in  contaminated  water, 
standard  methods69  have  been  devised  for  the  estimation  of  the 
bacterial  content  of  oysters.  These  are  similar  in  principle  and 
method  to  those  used  for  the  examination  of  water,  and  a  most 
important  index  of  sewage  contamination  and  consequent  danger 
of  typhoid  infection  is  the  number  of  colon  bacilli  present  in  the 
shell  fish.  The  shell  liquor  is  used  for  examination,  and  in  examining 
oysters  in  the  shell  the  following  procedure  is  followed :  Five  oysters 
having  deep  bowls  and  closed  shells  are  selected.  Lips  of  the  shell 
are  sterilized  in  the  flame  or  by  burning  with  alcohol.  The  liquor 
is  obtained  by  opening  the  shell  with  a  sterilized  knife,  or  better, 
by  drilling  a  hole  through  the  flame  surface  with  a  sterilized  gimlet. 
For  determining  the  total  number  of  bacteria  the  shell  liquor  is  with- 
drawn with  a  sterilized  pipette,  diluted  with  1  per  cent  salt  solution, 
and  placed  in  agar.  More  important,  however,  is  the  presumptive 
colon  test,  which  is  carried  out  by  inoculating  three  lactose  bile 
tubes  with  1.0  c.c.,  0.1  c.c.,  and  0.1  c.c.,  respectively,  from  each  of 
the  five  oysters.  The  tubes  are  incubated  for  three  days,  and  the 
development  of  over  10  per  cent  of  gas  in  the  closed  arm  is  con- 
sidered a  positive  reaction.  The  score  is  recorded  as  the  approximate 
number  of  colon  bacilli  contained  in  the  5.55  c.c.  of  shell  liquor 
from  the  five  oysters,  and  is  estimated  in  the  following  way:  A 
positive  reaction  in  a  tube  inoculated  in  1  c.c.  is  recorded  as  1.0, 
a  positive  reaction  in  0.1  c.c.  is  10,  and  in  0.01  is  recorded  as  100. 
The  sum  of  these  figures  is  the  score  for  the  batch  of  oysters  from 
which  the  five  have  been  taken.  In  examining  shucked  oysters  a 
well-mixed  sample  of  oysters  and  the  surrounding  fluid  are  put  in 
a  sterilized  vessel  and  lactose  bile  tubes  inoculated  in  triplicate 
with  1.0  c.c.,  0.1  c.c.,  0.01  c.c.,  0.001  c.c.  of  the  liquor.  No  definite 
standard  score  has  been  adopted,  but  the  United  States  Pure  Food 
Board  has  condemned  unshucked  stock  having  a  score  of  32  or 
higher. 


69Amer.  Jour.  Pub.  Health,  1913,  ii,  34. 


SECTION  VII 

PATHOGENIC  PROTOZOA 


FREDERICK  F.  RUSSELL,  M.D. 

INTKODUCTION 

IN  the  practice  of  his  profession  the  physician  requires  a  knowl- 
edge of  the  pathogenic  protozoa  found  in  man  and  the  domestic 
animals  and  of  their  closely  related  non-pathogenic  forms.  Quite 
commonly  in  the  diagnosis  of  fevers  it  is  necessary  to  examine  the 
blood  of  the  same  patient  for  both  malaria  and  bacteria,  therefore 
a  working  knowledge  of  the  principal  pathogenic  protozoa  is  essen- 
tial. In  this  work  it  will  be  possible  to  describe  the  forms  only  of 
medical  interest,  and  the  reader  is  referred  to  other  works  for 
further  information. 

The  protozoa  are  unicellular  animal  organisms  that  occur  singly 
or  in  temporary  colonies.  The  functions  of  the  animals  are  carried 
out  by  the  protoplasm  of  the  single  cell,  parts  of  which  may  be 
differentiated  for  special  purposes  and  are  then  called  organete. 


CLASSIFICATION    OF    THE    PROTOZOA 

CLASS  I.  SARCODINA  (Rhizopoda) . — The  body  is  naked  or  encased  and 
the  animal  moves  by  means  of  protruding  temporary  prolongations 
of  the  body  called  pseudopods.  They  possess  one  or  many  nuclei 
and  reproduce  by  fission  or  multiplication  in  a  cyst. 
Order  I.  Amcebce  (Lobosa). — Naked  or  with  a  simple  shell,  the 
pseudopia  are  lobose  or  finger-shaped,  the  nucleus  is  usually 
single  and  there  is  sometimes  a  contractile  vacuole.  Example, 
the  amcebce. 

CLASS    II.  MASTIGOPHORA    (Flagellata). — They    possess    flagella    for 
locomotion  and  for  obtaining  food;  they  may  be  naked  or  fur 

1048 


INTRODUCTION  1049 

nished  with  a  membrane ;  many  forms  possess  nucleus,  contractile 
vacuole  and  a  small  groove  spoken  of  as  the  cytostome.  Examples, 
the  trypanosomes  and  intestinal  flagellates. 

CLASS  III.  SPOROZOA. — They  live  parasitically  in  the  tissues  of  other 
animals,  ingesting  food  by  osmosis;  they  have  no  cilia  in  the 
adult  stage  but  may  form  pseudopoda,  one  or  more  nuclei,  no 
contractile  vacuole,  reproduction  by  spores.  They  are  divided  into 
two  subclasses,  telesporidia  and  neosporidia.  Examples,  gre- 
garinida,  coccidiidea,,  hemosporidia,  sarcosporidia,  etc. 

CLASS  IV.  INFUSORIA  (Ciliata). — The  body  is  generally  uniform  in 
shape,  with  cilia  and  contractile  vacuole,  and  usually  with  macro- 
and  micronucleus.  Examples,  pammecium,  balantidium. 


CHAPTER  LIV 

CLASS  I— SARCODINA   (RHIZOPODA) 
THE    AMOEBA 

THESE  organisms  belong  to  the  order  Amcebina  (Ehrenberg).  They 
are  characterized  during  the  vegetative  stage  by  a  semifluid  consist- 
ence, permitting  rapid  changes  of  form,  amoeboid  movements,  and 
progression  by  means  of  pseudopods.  There  is  no  internal  skeleton 
and  the  protoplasm  is  naked  and  may  be  differentiated  into  endo-  and 
ectoplasm,  and  in  some  cases  a  contractile  vacuole  is  present.  All 
forms  possess  one  or  more  nuclei.  Multiplication  takes  place  by  divi- 
sion into  two  or  more  daughter  cells.  Fertilization  possibly  takes  place 
by  the  conjugation  of  two  gametes. . 

Since  some  flagellates  possess  an  amoeboid  stage,  it  is  necessary  to 
know  most  of  the  life  cycle  of  an  organism  before  classifying  it  as  an 
ameba.  The  protoplasm  varies  greatly  in  its  consistency,  depending 
on  the  species  as  well  as  the  stage  of  the  life  cycle,  and  the  environ- 
ment and  food  supply.  Most  amoeba,  including  all  the  parasitic  forms 
(entamoebae),  possess  a  single  nucleus,  yet  Amoeba  diploidea  and  Amoeba 
binucleata  always  have  two,  and  the  other  species  may  show  more. 
The  nucleus  of  all  types  possesses  a  karyosome.  The  nucleus  is  well 
developed  and  in  it  may  be  followed  either  a  simple  or  typical  mitosis. 
The  cytoplasm  is  usually  at  some  stage  divided  into  a  granular  endo- 
plasm  and  a  clear  or  hyalin  ectoplasm,  the  latter  forming  the  pseu- 
dopods by  which  the  animal  moves  from  place  to  place. 

Until  recent  years  all  amoeboid  organisms  were  placed  in  the  genus 
Amoeba,  but  Schaudinn  established  a  genus,  the  Entamoeba,  for  the 
parasitic  species  which  have  many  points  of  difference  from  the  free 
living  varieties.  Of  the  free  living  forms,  the  easiest  to  study  is  the 
Amoeba  proteus  (Pallas),  a  very  large  organism,  200  microns  in 
diameter,  found  frequently  in  stagnant  water;  it,  however,  has  no 
direct  importance  in  medicine.  Another  group  of  free  living  amoeba) 
is  of  some  interest,  because  of  the  confusion  they  have  caused  in 
the  study  of  parasitic  amoeba;  they  are  the  so-called  "limax  amcrbse," 

1050 


SARCODINA 


1051 


which  have  been  cultivated  on  agar,  and  for  this  genus  Chatton  (1912) 
has  proposed  the  name  Valilkampfia.  They  are  small  organisms, 
5  to  30  microns  in  diameter,  provided  with  fingerlike  or  spinous  pseu- 
dopodia,  and  characterized  by  a  nucleus  witli  a  large  karyosome  and 
a  single  nucleated  resistance  cyst  in  which  no  multiplication  occurs. 
They  have  repeatedly  been  cultivated  from  human  dysenteric  stools, 
from  the  air,  and  apparently  from  liver  abscess  pus.  It  has  been 
shown,  beyond  doubt,  that  they  are  harmless  to  man,  and  that  they 
pass  through  the  intestinal  tract  with  food  and  water  in  the  cyst 


FIG.  120. — ENTAMBGEA  HISTOLYTICA.  Vegetative  form  showing  histolytica  type  of 
nucleus.  Stained  with  iron  hematoxylin.  (Army  Medical  School  Collection, 
Washington,  D.  C.) 

form.  While  they  will  develop  in  cultures  at  body  temperature,  a 
better  growth  is  obtained  at  the  temperature  of  the  room.  Since 
the  true  parasitic  amoeba?  have  never  been  cultivated  on  artificial 
media,  the  Vahlkampfia  may  be  dismissed  with  the  statement  that 
they  are  not  pathogenic. 

The  genus  Entamoebse  includes  all  human  parasitic  forms,  and  is 
characterized,  among  other  things,  by  the  absence  of  a  contractile 
vacuole,  which  is  always  present  in  Amo?ba  and  Vahlkampfia.  The 


1052  PATHOGENIC   PROTOZOA 

species  of  importance  to  physicians  are  Entamoeba  liistolytica,  En- 
tamceba  coli  and  Entamceba  gingivalis. 

Leidy  of  Philadelphia  established  the  genus  endamceba  which  may 
possibly  be  closely  related  to  or  identical  with  the  genus  entamocba, 
for  the  large  amoeba  which  is  parasitic  in  the  cockroach,  and  called 
it  Endamceba  blattce;  this  genus  presents ;  some  points  of  resemblance 
to  those  present  in  man,  but  its  life  history  has  not  been  sufficiently 
studied  by  our  present  methods  and  the  merging  of  the  two  at  this 
period  seems  scarcely  justifiable. 

The  nomenclature  of  the  human  parasitic  entamoebae  is  shown 
in  the  following  table  which  has  been  taken  from  Dobell  (1919)  : 


SYNOPSIS   OF  GENERA  AND   SPECIES   OF  AMCEB.^E  LIVING  IN 

j   MAN 

Genus  I.  ENTAMCEBA   (Casagrandi  and  Barbagallo,  1895. 

(nee  Endamoeba  Leidy,  1879.) 
Synonyms : 

Poneramceba  Liihe,  1908. 

•j  Chatton  and  Lalung-Bonnaire,  1912. 
Loschia      [ 

Proctambceba  Alexeieff,  1912 

(Amoeba  (pro  parte),  Endamceba,  Entameba,  Endameba,  En-, 
tamoba,  Auctt.) 

Type:  E.  coli  (Grassi)  Casagrandi  and  Barbagallo. 

Species  in  Man:  E.  coli  (Grassi)   Casagrandi  and  Barbagallo. 
E.  Jiistolytica  Schaudinn    (emend.  Walker). 
E.  gingivalis  (Gros)  Brumpt. 

Genus  II.  ENDOLIMAX  Kuenen  and  Swellengrebel,  1917. 

Only  species,  hence  type:  E.  nana  (Wenyon  and  O'Connor) 
Brug. 

Genus  III.  IODAMCEBA  nov.  gen. 

Only  species,  hence  type:  I.  butschlii  (Prowazek)  Dobell. 

Genus  IV.  DIENTAM(EBA  Jepps  and  Dobell,  1918. 

Only  species,  hence  type:  D.  fragilis  Jepps  and  Dobell, 


SARCODINA  1053 

ENTAMCEBA    HISTOLYTICA 

(EntamoBba  tetragena  [Viefeck],  Entamceba  africana  [Hartmann] 
Entamceba  nipponica  [Koidzumi,  pro  parte],  Entamoeba  tropicalis 
[Lesage,  pro  parte] ) 

It  has  long  been  customary  to  say  that  amoebse  as  a  cause  of 
disease  were  first  described  by  Lambl  of  Prague,  in  1860,  who  found 
them  present  in  the  stools  from  a  case  of  severe  diarrhea  in  a  child, 
but  some  zoologists,  Leuckart  (1863),  Grassi  (1888)  and  Dobell  (1919) 
believe  that  the  organisms  he  described  were  degenerated  trichomonads. 


FIG.  121. — ENTAMCEBA  HISTOLYTICA.    Vegetative  form,  simple  division.     (X  1300.) 
(Army  Medical  School  Collection,  Washington,  D.  C.) 

In  1870  Lewis  and  Cunningham  found  amoebae  in  20  per  cent  of  the 
stools  of  cholera  patients,  but  attached  no  pathogenic  importance  to 
them.  The  first  accurate  description  we  owe  to  Loesch  of  Petrograd, 
who  in  1875  studied  an  undoubted  case  of  amosbic  dysentery  with 
relapses,  and  he  named  the  organism  Amoeba  coli.  He  was  further 
successful  in  reproducing  the  disease  in  a  dog,  and  thus  began  its 
experimental  investigation.  Not  much  progress  was  made  until 
Kartulis  in  Egypt  began,  in  1886,  the  publication  of  a  long  series 
of  studies  which  has  continued  up  to  the  present  time,  and  because 
of  the  rich  clinical  and  pathological  material  at  his  disposal  his  work 
has  been  of  the  greatest  value.  In  1890  Osier  published  the  first  paper 


1054  PATHOGENIC   PROTOZOA 

in  America.  He  was  followed  by  Musser  and  Stengel  and,  in  1891, 
by  Dock,  and  Councilman  and  Lafleur.  The  work  of  the  last  two 
authors  was  especially  complete  and  firmly  established  the  entity  of 
this  disease  in  America.  In  1902  Jiirgens  differentiated  the  pathogenic 
amoeba  from  the  harmless,  and  in  1903  the  work  of  Schaudinn  ap- 
peared. This  author,  who  was  a  zoologist  by  training,  showed  clearly 
that  there  were  two  forms  of  parasitic  amoebae  and  he  followed  out 
most  of  the  details  in  their  life  history,  renaming  them  Entamoiba 
histolytica  and  Entamozba  coli.  Schaudinn  accepted  the  name  for 
the  genus  proposed  by  Casagrandi  and  Barbagallo  (1895)  but  named 
his  pathogenic  species  histolytica  and  the  non-pathogenic  coli.  These 
names  are  still  in  use  although  the  work  of  later  investigators  has 
shown  that  many  of  his  observations  were  erroneous.  Our  present 
knowledge  of  this  organism  we  owe  to  the  work  of  Craig,  Whitmore, 
Walker,  Sellards,  Darling,  Dobell  and  others. 


CLINICAL    DYSENTERY 

Dysentery  as  a  disease  has  been  known  from  the  earliest  times 
and  references  are  found  to  it  in  Sanscrit  and  Egyptian  literature 
and  in  early  Greek  and  Roman  writings.  Until  recent  years  its 
etiology  was  obscure,  but  we  now  recognize  two  separate  forms, 
bacillary  and  amoebic ;  the  former  has  already  been  described  under 
the  dysentery  bacilli.  Amoebic  dysentery  is  a  distinct  clinical  entity, 
and  runs  a  course  quite  different  from  the  bacillary  form.  It  begins 
gradually,  and  in  some  cases  is  chronic  in  character  from  the  start. 
Usually  there  is  no  rise  in  temperature  nor  any  great  change  in 
weight  or  health  until  the  disease  has  existed  some  time.  The  bowel 
movements  become  gradually  more  frequent  and  the  fecal  matter 
is  accompanied  by  larger  and  larger  amounts  of  mucus  and  blood. 
As  the  disease  progresses  and  more  and  more  of  the  colon  is  involved 
the  amount  of  blood  and  mucus  increases  until  the  stool  contains 
little  else.  The  colicky  pains  increase  in  frequency  and  severity 
and  there  is  added  tenesmus  and  finally  nausea  and  vomiting.  The 
patient  loses  flesh  and  strength  and  when  the  stools  increase  to 
twenty  and  thirty  daily,  becomes  bed-ridden.  The  abdomen  is  con- 
cave and  tender  on  pressure,  especially  over  the  colon.  The  course 
of  the  disease,  if  untreated,  tends  to  progress  with  periods  of  remis- 
sion, and  spontaneous  cure  probably  does  not  occur.  Bacillary 


SARCODINA  1055 

dysentery,  it  will  be  remembered,  is  a  disease  with  a  short  incuba- 
tion period  and  an  acute  onset;  after  two  or  three  days'  illness  the 
bacillary  ease  is  confined  to  bed,  is  pale,  weak,  emaciated  and 
presents  every  evidence  of  profound  toxemia ;  an  amoebic  case,  sick 
the  same  length  of  time,  will  be  up  and  about  and  perhaps  will  not 
have  applied  for  treatment. 

Complications. — A  common  and  most  dangerous  complication  is 
abscess  of  the  liver.  The  amoebae  travel  from  the  ulcers  in  the  colon 
by  way  of  the  lymphatics  to  the  liver  and  there  set  up  a  liquefying 
necrosis  of  the  parenchyma.  The  liquefied  portion  contains  a  red- 
dish or  chocolate-colored  fluid,  which  is  not  pus  in  the  ordinary- 
sense,  although  it  may  become  a  pus-containing  abscess  if  secondary 
bacterial  infection  occurs.  Liver  abscesses  may  be  single,  but  are 
much  more  often  multiple,  and  at  times  the  whole  liver  may  be 
riddled  with  large  and  small  abscess  cavities;  both  right  and  left 
lobes  may  be  involved.  If  surgical  interference  be  withheld,  the 
abscess  increases  in  size,  approaches  the  surface,  and  finally  ruptures 
into  the  lung  through  the  diaphragm  or  into  the  peritoneal  cavity. 
A  few  cases  of  amoebic  abscess  of  the  brain  have  been  reported. 
(Kartulis,  1904.) 

At  autopsy  the  lesions  are  found  in  the  colon,  principally  at  the 
sigmoid  flexure  and  in  the  cecum,  though  in  chronic  cases  the  whole 
colon  is  involved,  showing  ulcers  with  undermined  edges,  swollen 
solitary  follicles  and  a  hemorrhagic-catarrhal  inflammation  of  the 
mucous  membrane.  The  ulcers,  readily  differentiated  from  those 
caused  by  the  tubercle  bacillus,  are  of  all  sizes,  shallow  or  deep, 
and  are  characterized  by  irregular  margins  and  undermined  edges. 
Fresh  smears  made  at  autopsy  will  show  vegetative  amoebae.  In 
chronic  cases,  the  colon  is  a  mass  of  scars  and  ulcers  and  acutely 
inflamed,  swollen  and  thickened  mucous  membrane  resting  on  a 
hypertrophied  submucosa.  The  severe  and  chronic  forms  of  the 
disease  are  now  as  rare  as  they  were  formerly  common  as  a  result 
of  the  present  specific  treatment  with  emetin. 

Geographical  Distribution. — Although  amoebic  dysentery  is 
classed  among  the  tropical  diseases,  it  is  by  no  means  confined  to 
the  tropics.  In  the  United  States,  for  example,  it  is  endemic  as  far 
north  as  Baltimore  and  Washington,  and  cases  are  not  very  infre- 
quent in  the  northern  tier  of  states;  hence  one  must  examine  the 
stools  for  amoebae  in  dysenteric  cases  regardless  of  the  location  of 
the  patient's  home. 


1056  PATHOGENIC  PROTOZOA 

Diagnosis. — While  the  "history  of  the  case  may  suggest  amoebic 
infection,  the  diagnosis  can  only  be  made  with  certainty  by  micro- 
scopic examination  of  the  stool.  For  this  purpose  the  examination 
should  be  made  as  soon  after  the  stool  is  passed  as  possible;  and 
in  this  disease  it  is  usually  practicable  to  have  the  patient  come  to 
the  hospital,  clinic  or  office  and  pass  a  stool  there.  It  may  then  be 
examined  immediately.  If  this  is  impracticable,  the  stool  may  be 
kept  warm  and  sent  to  the  laboratory  in  a  small  glass  jar  inside 
a  tin  pail  partly  filled  with  water  at  body  heat;  a  little  cloth  or 
absorbent  cotton  will  hold  the  hot  water  and  prevent  splashing 
during  transit.  The  stool  will  show  bloody  mucous  masses,  and 
small  drops  of  this  are  placed  on  slides,  protected  with  a  cover  glass 
and  ringed  with  warm  vaseline  to  prevent  evaporation.  The  prepara- 
tion, to  be  of  value,  must  be  thin,  and  the  bloody  mucus  may  be 
diluted  with  salt  solution  if  necessary.  Except  in  hot  weather,  the 
slide  should  be  examined  on  a  warm  stage,  or  the  slide  may  be 
warmed  by  placing  heated  coins  on  it,  near  the  cover  glass.  At  least 
half  a  dozen  slides  should  be  examined  before  reporting  a  negative 
result.  Since  the  entamoebae  degenerate  and  die  soon  after  the  stool 
is  passed,  it  is  particularly  important,  when  studying  the  life  history, 
to  use  only  the  very  freshest  material.  Dobell1  believes  that  most 
of  the  mistakes  which  have  been  made  in  studying  the  life  history 
of  these  organisms  have  been  caused  by  the  examination  of  de- 
generated or  dead  parasites,  in  which  both  nucleus  and  cytoplasm 
may  have  been  abnormal. 

For  the  study  of  living  amoebae  and  cysts  it  is  helpful  to  mix 
a  particle  of  stool  in  a  drop  of  salt  solution  on  one  slide  and  in  a 
drop  of  iodine  solution  on  another.  (Iodine  should  be  used  as  a 
strong  aqueous  solution,  in  potassium  iodide— the  stronger  the  bet- 
ter. Dobell.)  The  iodine  penetrates  the  cyst  wall,  and  if  glycogen 
be  present  in  the  vacuoles,  gives  it  the  characteristic  color;  it  also 
acts  as  a  fixative  and  renders  the  nuclei  easily  visible,  so  that  they 
may  be  counted  and  the  details  of  structure  made  out  fairly  well. 

Stained  preparations  are  not  difficult  to  prepare,  although  the 
process  requires  some  time  and  care.  As  in  most  zoological  work, 
wet,  rather  than  dry,  fixation  is  used.  Thin  smears  are  made  on 
cover  glasses  or  slides  and  before  they  can  dry  are  covered  with 
or  immersed  in  Schaudinn's  fluid.  This  is  a  mixture  of  two  parts 

1  Dobell,  Clifford,  The  Amoebae  Living  in  Man,  London,  1919. 


SARCODINA 


1057 


of  a  saturated  solution  of  bichlorid  of  mercury  in  normal  salt 
solution  and  one  part  of  absolute  alcohol.  The  mercuric  solution 
is  prepared  by  adding  to  boiling  normal  salt  solution  a  little  more 
mercury  than  will  dissolve;  on  cooling,  some  of  the  bichlorid 
crystallizes  out.  At  no  stage  of  the  process  must  the  preparation 
become  dry  or  the  smear  is  worthless. 

1.  Fix  in  hot  (60°  C.)  Schaudinn's  fluid,  five  to  ten  minutes. 

2.  Harden  in  70  per  cent  alcohol  ten  to  thirty  minutes,  then  wash 
in  70  per  cent  alcohol  to  which  a  few  drops  of  tincture  of  iodin 
have  been  added  until  it  is  distinctly  colored — ten  minutes;  store 
in  70  or  80  per  cent  alcohol  until  ready  to  stain. 


FIG.   122. — ENTAMCBBA  HISTOLYTICA.    Motile  forms  showing  ingested  blood  cells 
and  clear  rectoplasm.     (Army  Medical  School  Collection,  Washington,  D.  C.) 

3.  The  Rosenbusch  hematoxylin  is  quite  satisfactory.     Transfer 
the  slides  to  distilled  water  and  change  several  times  until  they 
are  free  from  alcohol,  then  immerse  in  3.5  per  cent  iron-alum  solution 
for  from  half  an  hour  to  overnight. 

4.  Stain  in  the  following  solution,  after  rapid  washing  in  distilled 
water : 

(a)   1  per  cent  hematoxylin  in  95  per  cent  alcohol. 

(6)   Saturated  aqueous  solution  of  lithium  carbonate. 

Solution  (&)  is  added  to  solution  (a)  until  the  mixture  is  a  cherry 
red,  four  or  five  drops  of  lithium  to  10  c.c.-of  hematoxylin  is  sufficient. 

The  solution  is  either  pipetted  onto  the  slides  or  they  are  im- 
mersed in  it.  Stain  from  twenty  minutes  to  overnight. 

5.  Wash  thoroughly  in  distilled  water. 

6.  Differentiate  with  a  weak  iron-alum  solution   (three  parts  of 


1058  PATHOGENIC  PROTOZOA 

distilled  water  to  one  of  the  iron-alum  solution  is  satisfactory),  until 
the  slide  under  the  microscope  shows  the  structure  of  the  nucleus; 
the  examination  is  made  in  water  under  a  cover  glass. 

7.  When  the  differentiation  is  complete  the  slide  is  washed  in 
distilled  water  and  passed  through  graded  alcohols,  80,  95  and 
absolute  into  xylol  and  xylol-balsam.  This  stain  is  permanent. 

Romanowski  stains  on  dried  smears  may  be  used,  but  are  not  so 
good. 

In  fresh  specimens  Entamoeba  histolytica  presents  the  following 
appearance:  the  vegetative  forms  are  pale,  unstained  with  bile,  and 
are  seen  to  be  large  bodies,  20  to  30  microns  in  diameter,  consisting 
of  endo-  and  ectoplasm,  and  often  showing  a  delicate  nucleus  and 
also  many  inclusions  in  the  digestive  vacuoles,  principally  red  blood 
cells.  The  organisms  for  several  hours  after  the  stool  is  passed 
remain  actively  motile,  pushing  out  clear,  glass-like  pseudopods,  into 
which  the  granular  endoplasm  pours  as  the  amoeba  progresses  across 
the  field.  Even  when  there  is  no  progression  the  pseudopods  are 
protruded  or  retracted  first  in  one  then  in  another  direction.  There 
is  usually,  during  motion,  a  distinct  separation  of  the  clear  ectoplasm 
from  the  granular  endoplasm,  and  the  latter,  in  acute  cases  es- 
pecially, contains  many  red  blood  cells,  occasional  examples  show- 
ing as  many  as  twenty  or  thirty.  The  presence  of  red  blood  cells 
either  entire  or  partly  digested  is  characteristic  of  Entamoeba  his- 
tolytica. The  amoeba  is  sometimes  greenish,  and  it  is  supposed  that 
this  color  is  due  to  hemaglobin  liberated  from  the  ingested  red  cells. 
The  pseudopods  of  this  species  are  clear,  glassy  and  evidently  viscid 
and  dense  and  have  given  it  its  name  "histolytica,"  since  Schaudinn 
states  that  he  saw  the  amoeba  penetrate  the  mucous  membrane,  the 
pseudopods  dissecting  apart  the  epithelial  cells.  It  is  much  more 
probable  however,  that  the  parasite  secretes  a  strong  ferment,  which 
first  softens  and  then  dissolves  the  tissue  cells.  The  nucleus,  when 
the  endoplasm  is  packed  with  inclusions,  may  not  be  visible,  but 
further  search  will  reveal  amoebae  showing  a  nucleus.  It  is  vesicular, 
with  a  delicate  limiting  membrane,  and  as  it  is  highly  refractile, 
may  appear  as  a  clear  bright  spot.  As  the  specimen  grows  older 
the  amoebae  lose  much  of  their  motility  and  the  nucleus  may  become 
clearly  visible,  revealing  small  chromatic  dots  or  masses  adherent 
to  its  inner  surface  and  a  small  central  karyosomo. 

The  motile  amoebae  cannot  be  confused  with  anything  else,  but 
when  in  the  resting  stage  they  have  been  mistaken  for  swollen  and 


SARCOD1NA 


1059 


edcmatous  epithelial  cells.  A  little  attention  to  the  nucleus  will 
prevent  this  error,  since  the  tissue-cell  nucleus  is  large,  distinct,  and 
entirely  different  from  the  nucleus  of  an  amoeba. 

In  specimens  stained  with  hematoxylin  the  finer  details,  especially 
in  the  nucleus,  may  be  studied,  but  stained  preparations  are  never 
necessary  for  clinical  diagnosis.  In  smears  from  fresh  cases  vegeta- 
tive forms  only  are  found,  later  many  degenerative  forms  appear 
and  during  convalescence  only  cysts  may  be  seen.  In  stained  speci- 
mens there  is  rarely  any  separation  of  ectoplasm  and  endoplasm, 
but  the  nucleus  is  always  visible.  The  cytoplasm  is  granular  and 
has  a  coarse  honey-combed  appearance.  The  nucleus  shows  a  dis- 


FIG.  123.  —  ENTAMCEBA  HISTOLYTICA. 
(Army  Med.  School  Collection,  Wash- 
ington, D.  Cj 


FIG.  124. — ENTAMCEBA  HISTOLYTIC.— 
(X  1150)  Cyst,  showing  four  nuclei, 
two  of  which  are  very  distinct,  and 
large  chromatoid  body.  (Army  Med. 
School  Collection, Washington,  D.  C.) 


tinct,  though  delicate,  limiting  membrane,  on  the  inner  surface  of 
which  are  few  or  many  chromatin  dots.  In  the  center  is  a  small 
karyosome,  which  may  show  a  central  body  or  centriole.  The  outer 
zone  of  the  nucleus  has  a  honey-comb  structure,  in  which  are  im- 
bedded granules  of  chromatin. 

Multiplication  in  the  vegetative  stage  is  by  division  into  two 
daughter  cells ;  in  the  intestinal  contents,  it  is  difficult  to  find  forms 
which  are  undergoing  division,  but  a  few  have  been  described 
and  pictured.  In  experimental  dysentery,  in  the  cat,  however,  all 
stages  of  the  process  may  be  followed  by  removing  the  intestine 


1060  PATHOGENIC  PROTOZOA 

from  an  infected  animal  which  has  been  killed  and  making  serial 
sections  of  the  ulcers.  In  such  sections  it  can  be  seen  that  the 
chromatin  of  the  nucleus  migrates  from  the  nuclear  membrane  to- 
ward the  center,  and  the  nucleus  elongates,  being  first  oval  and 
later  spindle  shaped;  although  the  chromatin  dots  and  threads  and 
the  achromatic  fibers  can  be  readily  seen  in  the  well  stained  speci- 
mens they  do  not  show  the  usual  typical  figures  of  a  typical  mitosis, 
but  are  arranged  in  an  atypical  and  irregular  manner.  The  elongated 
nucleus  becomes  constricted  in  the  middle  and  a  little  later  divides 
and  the  cytoplasm  soon  follows,  leaving  two  daughter  cells. 

Degenerative  Forms. — These  are  extremely  common  in  stale 
stools,  in  cases  during  convalescence,  or  under  active  treatment,  and 
also  in  experimental  dysentery  in  the  cat,  and  they  have  led  to 
much  confusion  in  the  past.  The  nucleus  breaks  up  into  fragments 
and  chromatin  masses  are  extruded  into  the  cytoplasm  in  irregular 
forms,  and  parts  of  the  cells  are  apparently  budded  off.  At  one 
time  the  budding  process  was  looked  upon  as  normal  by  Schaudinn 
and  his  followers,  but  there  is  now  little  doubt  that  both  spores 
and  buds  are  degenerative  changes  and  that  the  animal  multiplies 
only  by  binary  fission  in  the  vegetative  forms  or  by  the  development 
of  four  nuclei  in  the  cysis. 

Cyst  Formation. — The  encystment  follows  the  general  rule  in 
that  under  suitable  conditions,  an  amoeba  comes  to  rest,  ejects  all 
food  particles  from  the  cytoplasm  which  becomes  finally  granular, 
and  round,  and  then  secretes  a  cyst  wall,  and  in  this  condition 
•passes  out  of  the  body  with  the  feces.  The  cyst  of  Entamoeba  his- 
tolytica  was  first  described  by  Quincke  and  Roos  (1903),  and  again 
by  Huber  (1903),  but  without  making  any  real  impression  on  the 
medical  or  zoological  opinion  of  the  day.  They  were  redis- 
covered by  Viereck  (1907)  and  called  by  him  Entamoeba  tetra- 
gena,  and  for  a  time  was  believed  to  be  a  new  species.  In  fact,  Hart- 
mann  described  a  vegetative  stage  of  Entamoeba  tetragena  as  Ent- 
amoeba africana,  afterwards  accepting  the  name  "tetragena,"  but 
it  is  now  apparent  that  tetragena  is  merely  the  end,  or  cyst  stage, 
of  Entamceba  histolytica,  which  had  formerly  been  overlooked  by 
Schaudinn  and  his  followers.  Cysts  are  not  easily  found  in  all  cases, 
and  it  is  possible  that  when  treatment  is  vigorous  they  never  develop. 
They  are,  without  doubt,  the  form  in  which  'the  parasite  leaves  the 
body  to  infect  new  victims;  because  of  their  heavy  cyst  wall  they 
are  quite  resistant.  The  protoplasm  of  the  cyst  and  the  precystic 


SARCODINA  1061 

stage  is  granular,  but  shows  no  vacuoles  nor  cell  inclusions.  The 
nucleus  undergoes  division  by  mitosis  first  into  two,  and  then  four 
small  ring-like  nuclei,  and  the  presence  of  these  four  nucleated 
cysts  is  pathognomonic  of  the  disease.  They  may  be  found  most 
abundantly,  not  in  the  small  amount  of  mucus  which  may  adhere 
to  the  formed  feces,  but  in  surface  scrapings  from  the  fecal  mass. 
In  addition  to  the  four  small  ring-like  nuclei,  the  cysts  contain  few 
or  many  clumps  of  chromatin;  these  in  total  mass  may  be  many 
times  greater  than  the  nucleus,  and  it  is  impossible,  therefore,  that 


FIG.  125. — ENTAMGEBA  COLI.     (Army  Merl.  School  Collection,  Washington,  D.  C.) 

they  are  simply  extruded  from  the  nucleus ;  evidently,  the  chromatin 
grains,  while  in  the  cytoplasm,  increase  in  size  and  number.  In 
hematoxylin  stains  no  structure  in  these  masses  is  discernible  and 
their  function  is  unknown ;  it  is  possible  that  they  are  merely  reserve 
food  material;  after  a  time  they  disappear  and  one  finds  cysts  quite 
free  of  them.  The  presence,  however,  of  many  large  chromatin 
masses  in  the  cysts  is  quite  characteristic  of  Entamoeba  histolytica. 
These  masses,  which  stain  deeply  with  iron-haematoxylin,  have  been 
given  various  names:  chromatin,  chromatoid  masses,  chromedia, 
crystalloids,  inclusions,  etc. 


1062 


PATHOGENIC   PROTOZOA 


Fertilization  inside  the  cyst  has  not  been  demonstrated,  nor  has 
any  other  sexual  process  or  conjugation  been  shown  to  occur. 

In  size  the  cysts  from  different  patients  vary  considerably,  so 
much  so  that  well  recognized  races  or  pure  lines  occur :  Dobell  and 
Jepps  (1918)  describe  five  such  races,  the  cysts  of  which  have 
average  diameters  of  6.6  microns,  8.3  microns,  11.6  microns,  13.3 
microns  and  15.0  microns. 

The  Dysentery  Carrier. — Both  convalescent  and  healthy  contact 


FIG.  126. — ENTAMCEBA  COLI. 


Typical  nucleus.     (Army  Med.  School  Collection, 
Washington,  D.  C.) 


carriers  are  known,  and  recent  experiments  have  shown  that  they 
are  not  infrequent,  even  in  the  absence  of  cases  of  chemical  dysen- 
tery. To  explain  the  carrier  state,  it  is  of  course  necessary  to 
predicate  some  insignificant  and  silent  lesions  in  the  colon  of  the 
apparently  healthy  man.  There  the  first  part  of  the  life  history 
is  lived  through  and  the  parasites  which  come  to  lie  in  the  lumen 
of  the  intestine  encysts  and  are  found  in  the  feces. 

The  treatment  of  amoebic  dysentery,  to  be  effective,  must  be  radical 
and  persistent,  and  may  be  compared  to  the  treatment  of  malaria 
with  quinine.  For  many  years  the  English  in  India,  with  a  few 


SARCODINA  1063 

followers  in  other  parts  of  the  world,  had  treated  dysentery  with 
ipecac  in  massive  doses,  with  wonderful  results  in  some  cases  and 
failure  in  others.  The  treatment  was  quite  disagreeable  and  not 
entirely  satisfactory.  Vedder,  in  1911,  examined  the  various  al- 
kaloids of  ipecac  and  found  that  emetin  was  strongly  amoebacidal, 
and  he  recommended  its  use  for  this  disease.  Rogers,  in  India, 
following  out  this  suggestion,  soon  reported  excellent  results,  and 
the  drug  is  now  accepted  as  a  specific.  It  is  administered  hypo- 
dermically  in  1/3-grain  doses  three  times  a  day  at  first,  then  twice 
and  later  once  daily  until  a  total  of  ten  grains  has  been  administered 
(Vedder).  In  addition,  the  patient  is  put  to  bed  and  placed  on  a 
milk  diet.  During  convalescence  large  doses  of  bismuth  subnitrate, 
a  heaping  teaspoonful  suspended  in  water  or  milk,  may  be  given 
(Decks).  Relapses  are  of  course  treated  in  the  same  manner  as 
primary  infections.  As  a  result  of  the  emetin  treatment  and  exact 
diagnosis  the  clinical  picture  of  amoebic  dysentery  has  completely 
changed,  and  we  no  longer  see  the  weak  and  emaciated  dysenteries 
who  formerly  crowded  the  wards  of  tropical  hospitals. 

Epidemiology. — One  significant  fact  appears  in  the  epidemiology 
of  the  disease — it  always  occurs  sporadically  and  never  in  explosive 
epidemics  such  as  we  see  in  water-borne  diseases,  like  typhoid  and 
cholera ;  house  epidemics  are,  on  the  contrary,  not  uncommon.  This 
fact  points  to  the  importance  of  contact;  and  flies,  as  the  chief 
agents  in  its  spread.  Buxton2  and  others  have  examined  the  drop- 
pings and  intestinal  contents  of  flies  caught  in  latrines.  Buxton 
found  0.3  per  cent  of  a  thousand  flies  harboring  E.  histolytica  cysts, 
so  that  there  remains  little  doubt  but  that  the  house  fly  is  one  of 
the  principal  carriers  of  dysentery.  Extreme  cleanliness  among  the 
servants  and  in  the  kitchen  will  prevent  the  transfer  of  histolytica 
cysts  from  the  ill  to  the  well.  The  disease  has  disappeared  from  the 
Panama  Canal  Zone,  where  it  formerly  was  common,  since  the  intro- 
duction of  good  water  and  sewer  systems  and  better  hygienic 
conditions. 

ENTAMffiBA  COLI  (grass!  1879)  casa  grand!  et  Barbagallo  1895 

This  is  a  harmless  parasite  of  man,  and  its  presence  in  stools,  at 
one  time,  gave  rise  to  much  confusion,  and  in  the  minds  of  many, 


2  Buxton,  P.  A.,  The  Importance  of  the  House  Fly  as  a  Carrier  of  E.  histoly- 
tica.    Brit.  Mecl.  Jour.,  London,   1920,   142. 


1064  PATHOGENIC   PROTOZOA 

threw  doubt  upon  the  existence  of  a  form  of  dysentery  due  to  amoeba, 
since  it  was  found  not  infrequently  in  healthy  individuals.  Schau- 
dinn  found  it  present  in  the  stools  of  50  per  cent  of  the  persons 
examined  in  East  Prussia,  in  Berlin  in  20  per  cent,  and  in  Istria 
in  60  per  cent.  Craig,  and  Craig  and  Ashburn  found  it  present  in 
176,  or  58  per  cent,  of  307  examinations  of  healthy  American  soldiers. 
Craig  was  able  to  follow  some  individuals  for  four  to  six  years, 
during  which  time  they  constantly  showed  Entamoeba  coli  in  the 
feces,  yet  never  developed  dysentery.  The  organism  seems  to  be 


FIG.  127. — ENTAMCEBA  COLI.     Small  precystic  form.      (Army  Med.  School  Collec- 
tion, Washington,  D.  C.) 

found  iii  all  countries,  regardless  of  climate.  Its  recognition  and 
separation  from  histolytica  we  owe  to  Schaudinn,  Jurgcns,  Craig 
and  others. 

In  size  it  varies  from  ten  to  forty  microns,  the  average  being 
between  twenty  and  thirty.  The  ecto'plasm  is  never  seen  except 
during  movement,  and  it  is  then  hyaline,  and  only  slightly  refractile, 
and  much  more  fluid  than  in  histolytica.  The  digestive  vacuoles 
rarely,  if  ever,  contain  red  blood  cells,  but  are  filled  with  cocci  and 
bacilli,  a  form  of  food  not  seen  in  healthy  histolytica.  In  general, 
the  vacuoles  are  larger  and  more  numerous  in  coli  than  in  histolytica, 
and  the  motility  is  feebler.  In  fresh  specimens  the  nucleus  is  rather 


SARCODINA  1065 

easier  to  find  than  in  histolytica,  and  is  distinctly  outlined  by  a 
heavy,  double-contoured  membrane.  The  nucleus,  as  in  all  amoeba, 
is  vesicular,  and  shows  a  small  eccentric  karyosome  and  dots  of 
chromatin  on  the  nuclear  membrane  and  imbedded  in  the  nuclear 
network. 

Multiplication  in  the  vegetative  stage  is  by  binary  fission  of  the 
nucleus  and  the  cytoplasm,  resulting  in  two  daughter  cells. 


FIG.  128. — ENTAMCEBA  COLI.     CYST  showing  a  large  vacuole.      (Army  Med.  School 
Collection,  Washington,  D.  C.) 

Cyst  Formation. — This  is  characteristic  of  the  species,  and  it 
furnished  one  of  the  principal  reasons  for  the  separation  of  coli 
and  histolytica.  Before  encysting  the  animal  frees  itself  of  all 
inclusions  and  becomes  clear,  transparent,  and  assumes  a  spherical 
form,  and  secretes  a  cyst  wall.  The  nucleus  divides  first  into  two, 
then  four,  and  finally  eight  daughter  nuclei ;  there  is  a  large  vacuole 
containing  glycogen  which  reaches  its  maximum  size  in  the  double 
nucleus  stage ;  it  later  disappears  and  is  not  seen  in  the  mature  cyst. 
Schaudinn  described  a  complicated  autogamy  in  the  cyst,  yet  later 
researches  by  Hartmann  and  Whitmore  show  nothing  more  than 


1066  PATHOGENIC  PROTOZOA 

repeated  binary  division  of  the  nucleus.  The  normal  number  of 
nuclei  in  a  coli  cyst  is  eight,  yet  occasionally  cysts  are  seen  in 
which  division  has  gone  on  until  there  are  as  many  as  sixteen.  In 
size  the  cyst  measures  10  to  30/x  or  more. 

Cats  or  human  beings  may  be  parasitized  by  feeding  material 
containing  coli  cysts,  and  in  nature,  as  the  cysts  are  the  resistant 
forms  of  the  parasite,  the  infection  is  probably  transmitted  from 
one  host  to  another  by  means  of  them.  No  disease,  however,  results, 
though  the  amoebae  continue  to  be  present  in  the  stools  for  years. 


FIG.   129.— ENTAMCBBA  COLI.     Cyst  showing  eight  nuclei.      (Arch,  fur  Protisten- 

kunde,  1912,  xxiv.) 

•  It  is  possible  that  fertilization  takes  place  between  the  young 
amoebae  (gametes?),  which  are  liberated  when  the  cyst  dissolves  in 
u  new  host,  as  is  the  case  with  Entamoeba  blattas. 


ENTAMCEBA    GINGIVALIS 

(Gros  1849,  emend,  von  Prowazek  1904) 

This  amoeba  is  found  in  the  human  mouth  both  in  health  and 
disease.  It  has  been  described  at  different  times  under  various  names 
(bucalis,  dentalis)  by  Gros,  Steinberg,  von  Prowazek,  Lewald,  Smith 
and  Barrett,  Chiavaro  and  Craig,  and  quite  recently  has  been  sug- 
gested as  the  cause  of  pyorrhea  alveolaris  by  Smith  and  Barrett  and 
Bass  and  Johns.  It  is  widely  distributed,  and  has  been  reported  from 
all  quarters  of  the  world. 

The  organism  is  easily  found  in  the  tartar  at  the  base  of  the 
teeth,  in  cavities  in  the  teeth,  and  even  at  the  gum  margin  in  healthy 
mouths.  It  varies  in  size  from  seven  to  thirty-five  microns,  averaging 


SARCODINA  1067 

between  twelve  and  twenty  (Craig).  Motility  is  well  marked,  though 
it  is  not  so  active  an  organism  as  histolytica,  the  pseudopods,  mostly 
short  and  blunt,  being  formed  of  the  clear,  slightly  refractile 
ectoplasm.  The  endoplasm  is  granular,  contains  the  nucleus  and  many 
food  vacuoles  containing  nuclei  of  leucocytes  and  granular  matter, 
and  possibly  a  few  red  blood  cells.  Although  Dobell  and  others  main- 
tain that  red  cells  are  never  found.  The  nucleus  is  small,  and  in  fresh 
specimens  is  usually  invisible.  In  structure,  it  is  not  unlike  the 
nuclei  of  coli  and  histolytica;  the  chromatin  granules  form  a  compact 
and  distinct  ring  obscuring  the  nuclear  membrane.  The  karyosome 
is  small  but  distinct  and  is  either  central  or  slightly  excentric  and 
is  surrounded  by  a  clear  achromatic  halo.  There  is  no  chromatin 
visible  between  the  karyosome  and  the  nuclear  margin. 

Before  encysting  the  parasites  are  much  reduced  in  size,  and  the 
cytoplasm  frees  itself  from  all  inclusions  and  becomes  clear,  spherical 
and  immobile.  The  cysts  are  small,  eight  to  ten  microns,  circular 
and  definitely  outlined,  sometimes  with  a  double  contour,  and  in 
stained  specimens  the  nucleus  is  always  visible.  It  is  small,  averaging 
only  three  microns  (Craig),  making  it  smaller  than  in  histolytica 
and  coli.  The  limiting  membrane,  while  not  heavy,  is  distinct,  and 
encloses  a  nuclear  body  having  very  little  chromatin  other  than  the 
small  centrally  located  karyosome.  Multiplication  occurs  only  in 
the  vegetative  state  and  by  binary  fission. 

Cyst  Formation. — Cysts  are  rarely  observed,  and  then  in  small 
numbers ;  the  cyst  wall  is  not  heavy,  but  may  show  a  double  contour ; 
the  protoplasm  is  clear,  free  from  all  inclusions  and  vacuoles  and 
shows  a  single  small  nucleus,  but  without  any  signs  of  multiplication. 
It  is  apparent,  therefore,  that  the  cyst  is  a  protective  stage  and 
has  nothing  to  do  with  reproduction,  which  occurs  in  the  vegetative 
state  only.  In  this  respect  it  resembles  the  Vahlkampfia.  The  find- 
ing of  cysts  is  so  rare  that  many  investigators  doubt  their  existence 
altogether,  and  suggest  that  cysts  when  found  may  pertain  to  some 
other  organism  accidentally  present  in  the  mouth.  Further  studies 
are  necessary. 

Transmission  of  the  infection  could  occur  directly  by  contact 
from  person  to  person  by  kissing,  and  a  cyst  stage  is  not,  .therefore, 
essential  to  the  survival  of  the  parasite. 

Although  the  organism  is  almost  constantly  present  in  pyorrhea 
alveolavis  it  is  also  found  in  healthy  mouths,  and  in  the  absence  of 
all  experimental  proof,  it  is  doubtful  if  the  organism  is  of  patholog- 


1068  PATHOGENIC   PROTOZOA 

ical  importance.  Emetin  has  a  decided  effect  upon  many  cases  of 
pyorrhea  alveolaris,  and  under  that  treatment  alone  the  disease 
may  disappear ;  the  nature  of  its  therapeutic  action  is  not  yet  clear, 
and  does  not  necessarily  indicate  any  etiological  relationship. 


ENDOLIMAX  NANA  (Wenyon  and  O'Connor,  1917)  Burg,  1918 

(Amoeba  Umax,  Wenyon,  1916.  Entamceba  nana,  Wenyon  and  O'Con- 
nor, 1917.  Endolimax  intestinal™,  Kuenen  and  Swellengrebel, 
1917.) 

This  organism  was  first  described  in  1917  by  Wenyon  and  0  'Con- 
nor, by  Swellengrebel  and  Mongkoe  Winoto,  by  Kuenen  and  Swel- 
lengrebel and  by  Dobell  and  Jepps.  The  last  mentioned  authors 
believe  it  to  be  the  commonest  inhabitant  of  the  human  bowel.  It 
is  agreed  by  all  investigators  that  it  is  not  pathogenic  and  that 
its  principal  importance  is  due  to  the  possibility  of  confusing  it 
with  E.  histolytica.  In  the  vegetative  stage  it  is  quite  small,  usually 
measuring  6  to  12  microns  in  diameter.  It  usually  contains  food 
vacuoles  filled  with  bacteria  but  no  blood  cells  and  never  contractile 
vacuoles.  Its  movements  are  sluggish,  resembling  those  of  E.  coli, 
the  differentiation  between  ecto-  and  endoplasm  is  not  clear  cut 
and  the  pseudopods  are  few  and  blunt.  Outside  the  body  all  move- 
ment soon  ceases. 

The  nucleus  is  seen  with  difficulty  in  the  living  specimen  but 
when  the  organisms  are  properly  fixed  and  stained  with  iron 
haematoxylin  the  nucleus  becomes  the  most  characteristic  feature 
of  the  parasite.  It  is  vesicular  and  1  to  3  microns  in  diameter 
averaging  between  2  and  2.5  microns.  The  karyosome  is  large  and 
pleomorphic.  It  consists  of  a  principal  mass,  which  is  excentrically 
located  and  provided  with  few  or  many  lobes,  which  are  often 
almost  detached  from  the  main  body,  being  connected  only  by  a 
narrow  isthmus.  It  is  necessary  to  have  well  differentiated  stains" 
or  the  details  of  structure  will  be  overlooked. 

If  the  stool  is  not  fresh  the  amoebae  may  degenerate  and  the 
nucleus  present  quite  a  different  appearance;  the  segments  of  the 
karyosome  can  no  longer  be  distinguished  and  the  total  mass  may 
come  to  lie  on  the  outer  ring  of  cliromatin  granules,  giving  rise 
to  a  signet  ring  appearance.  It  is  best  therefore  to  obtain  the  fresh- 
est possible  material  for  study, 


SARCODINA  10G9 

Cysts. — Before  encystment  the  parasite  extrudes  all  food  par- 
ticles and  the  protoplasm  becomes  clear,  all  movement  ceases  and 
the  parasite  assumes  a  round  or  oval  form  and  the  secretion  of  a 
cyst  wall  begins.  The  mature  cyst  has  four  nuclei,  whose  internal 
structure  resembles  that  of  the  vegetative  form;  that  is,  there  is  a 
karyosome  made  up  of  a  central  mass  and  more  or  less  detached 
lobules  of  chromatin.  There  are  no  chromatoid  rods  in  the  cytoplasm 
as  in  histolytica  but  there  are  granules  which  give  the  staining 
reactions  of  volutin.  In  iodin  solution  the  cysts  are  stained  yellow ; 
in  some  may  be  noted  masses  of  glycogen,  but  this  substance  is 


a  b  c  d 

FIG.  130. — ENDOLIMAX  NANA.  This  figure  contains  figures  Nos.  23,  25,  26  and  27  of 
plate  No.  2,  Dobell's  "Amoebae  Living  in  Man."  The  first  figure  on  the  left 
represents  an  active  amoeboid  form.  The  next  three  show  uninucleate,  binu- 
cleate  and  quadrinucleate  (mature)  cysts,  respectively.  Plate  11  of  "Amoebae 
of  Living  Man"  by  Clifford  Dobell,  published  by  John  Bale,  Sons  &  Danielsson, 
London. 

commoner  in  the  vegetative,  precystic  and  young  cysts  than  in  those 
which  are  mature. 

Pathogenicity. — It  is  not  believed  that  the  parasite  causes  any 
disease ;  it  may  however  be  found  in  connection  with  histolytica  and 
thus  cause  confusion.  No  treatment  so  far  given  has  had  any 
influence  on  the  organism. 


IODAMCEBA  BUTSCHLII  (Prowazek,  1912,  Emend.  Dobell,  1919) 

(Entamceba    butschlii,   Prowazek,    1912.      Iodin   Cysts   or   I.    Cysts, 
Wenyon,  1916.    Pseudolimax,  Kuenen  and  S\v  ellengrebel,  1917.) 

"Wenyon  in  1915  described  quite  briefly  some  spherical  bodies 
containing  an  inclusion  which  stained  with  iodin  solutions.  Later 
he  called  these  I  or  iodin  cysts  and  under  that  name  they  have 
been  recognized  by  many  as  not  very  rare.  They  are  always  found 


1070  PATHOGENIC   PROTOZOA 

in  connection  with  some  other  amoeba  and  never  alone,  but  it  is 
not  believed  that  they  have  any  pathological  importance. 

Until  recently  the  vegetative  stage  had  not  been  recognized,  but 
in  1919  Dobell  published  a  complete  description,  and  identified  the 
parasite  with  the  one  incompletely  described  by  Prowazek  as  En- 
tamoeba  biitschlii  in  1912. 

In  size  the  organism  is  small,  averaging  9  to  13  microns,  and  it 
resembles  E.  coli  in  general  appearance  except  that  the  nucleus  is 
almost  if  not  quite  invisible,  particularly  in  organisms  with  food 
vacuoles  filled  with  bacteria  and  granular  matter. 

In  stained  specimens  the  structure  of  the  nucleus  serves  to  dis- 
tinguish it  from  other  intestinal  amoebae.  The  nucleus  is  vesicular 
and  contains  a  relatively  large  karyosome,  one-third  to  one-half  the 
diameter  of  the  nucleus.  Between  the  karyosome  and  the  well 
developed  nuclear  membrane  lies  a  row  of  granules  of  peripheral 
chromatin  which  Dobell  has  succeeded  in  counterstaining  with  eosin 
in  well  differentiated  hematoxlyn  preparations.  It  is  the  presence 
of  this  large  karyosome  and  the  layer  of  peripheral  chromatin  dots 
that  permit  the  differentiation  of  the  organism  from  E.  coli;  the 
two  organisms  are  alike  in  their  food  habits. 

The  cysts  are  peculiar  and  quite  unlike  those  of  any  other 
intestinal  amcebas.  In  shape  they  are  irregular  although  some  are 
round  or  oval.  In  measuring  them  Dobell  averaged  the  two  prin- 
cipal dimensions  and  found  that  the  average  size  is  9  or  10  microns, 
with  extreme  of  6  to  16  microns.  The  nucleus  of  the  cysts  is  single, 
and  is  distinguished  by  having  the  karyosome,  which  has  already 
been  described,  pass  to  the  periphery  and  come  to  rest  against  the 
nuclear  membrane,  giving  rise  to  a  well  marked  signet  ring  appear- 
ance. Peripheral  granules  of  chromatin  may  still  be  made  out  in 
well  stained  specimens.  Occasional  cysts  may  have  two  nuclei  but 
they  may  be  interpreted  in  the  same  way  that  we  interpret  more 
than  the  usual  number  of  nuclei  in  E.  histolytica  and  coli,  as  an 
abnormality. 

The  iodin  masses  in  the  cysts  which  give  this  organism  its  name 
are  well  brought  out  by  staining  the  fresh  specimens  in  iodin  solu- 
tion. As  a  rule  the  glycogen  mass  is  single,  large  and  has  well 
defined  borders,  but  its  appearance  varies  with  its  age ;  the  precystic 
amoeba  shows  merely  a  diffuse  brown  stain,  older  specimens  appear 
as  above,  although  occasional  specimens  may  show  more  than  one 
iodin  staining  mass.  Throughout  the  cytoplasm  may  be  seen  granules 


SAUCODINA 


1071 


of  volutin  and  the  granules  increase  in  visability  as  encystment 
proceeds. 

The  cysts,  like  those  of  other  amoebae,  do  not  withstand  drying 
but  they  remain  alive  in  feces  or  water  for  two  or  three  weeks 
without  undergoing  any  further  development. 


35 


37 


39  40  41  42 

FIG.  131. — IODAMCEBA  BuTSCHLii.  Taken  from  Dobell  as  above.  The  numerals 
on  the  plate  are  Dobell's  figure  numbers.  Nos.  35  and  36  represent  precystic 
amoebae.  Observe  changes  taking  place  in  nuclear  structure,  freedom  from 
cytoplasmic  inclusions,  etc.  No.  37  is  an  organism  just  encystic,  volutin 
granules,  pink  in  cytoplasm,  small  clear  space  representing  glycogen.  No.  39 
is  a  mature  cyst,  large  red  glycogen  mass.  Nos.  40,  41  and  42  are  mature  cysts 
showing  typical  structure  of  nucleus,  volutin  granules  and  glycogen  vacuole. 
Nos.  41  and  42  are  cysts  of  irregular  shape  often  formed  by  this  species  and  not 
artifacts.  Plate  11  of  " Amoebae  of  Living  Man"  by  Clifford  Dobell,  published 
by  John  Bale,  Sons  &  Danielsson,  London. 

This  parasite  has  been  found  in  the  healthy  as  well  as  in  dysen- 
tery cases  and  there  is  no  evidence  that  it  has  any  pathogenic 
power,  it  disappeared  under  the  administration  of  cmctin  and  in 
this  point  alone  resembles  E.  histolytica. 


1072  PATHOGENIC  PROTOZOA 


DIENTAMCEBA  FRAGILIS  (Jepps  and  Dobell,  1918) 

This  is  the  only  species  of  the  genus  and  was  discovered  and 
described  by  Dobell  and  Jepps  in  1918,  although  it  is  probable  that 
Wenyon  saw  but  did  not  describe  it  in  1909. 

Only  eight  cases  of  proved  infection  are  known,  although  it  is 
probable  that  it  is  commoner  than  this  would  lead  one  to  suppose 
since  the  organism  dies  quickly  and  the  degenerated  forms  are 
soon  unrecognizable.  It  is  quite  small  the  average  size  being  8  or  9 
microns  with  extremes  of  3.5  to  12.  The  eeto-  and  endoplasm  is 
sharply  differentiated  and  the  pseudopods,  consist  almost  entirely  of 
ectoplasm.  The  cytoplasm  contains  food  vacuoles  filled  with  bacteria 
but  no  red  cells  are  ever  seen;  the  organism  is  probably,  therefore, 
a  pure  saprophyte  and  without  pathological  importance. 

The  nucleus  is  characteristic  and  is  typically  double,  although  a 
few  examples  with  only  one  nucleus  have  been  seen.  The  nucleus 
varies  in  size  with  the  size  of  the  cell;  its  nuclear  membrane  is 
delicate  and  free  from  chromatin  particles.  All  the  chromatin  of 
the  nucleus  is  accumulated  in,  the  central  karyosome  where  it  consists 
of  a  number  of  granules  of  varying  distinctness  all  imbedded  in  a 
linin  network.  The  discoverers  believe  that  the  cell  grown  to  full 
size  and  then  divides  into  two  uninucleate  daughter  cells,  which 
increase  in  size  and  the  single  nucleus  divides  into  two.  Up  to  the 
present  time  no  cysts  have  been  found,  and  in  this  it  possibly 
resembles  E.  gingivalis,  in  which  the  presence  of  cysts  is  still  doubtful. 


CHAPTER  LV 

CLASS  II— MASTIGOPHOKA   (DIESING) 
SUB-CLASS— FL  AGE  LL  AT  A   (COHN  EMEND.  BUTSCHLI) 

ORDER    I— POLYMASTIGINA    (Blochmann) 

THESE  are  flagellates,  possessing  three  to  eight  flagella. 

Technic  of  the  examination  for  intestinal  flagellates:  It  is  not 
necessary  to  administer  a  cathartic  unless  motile  vegetative  forms  are 
desired;  cysts  are  found  in  formed  stools.  Small  particles  of  feces 
are  mixed  with  a  drop  of  water,  or  dilute  stain  and  the  slide  is 
examined  with  a  high,  dry  lens,  after  a  cover  glass  has  been  applied. 
The  stains  most  used  are  Gram's  iodin  and  dilute  eosin;  the  iodin 
besides  staining  the  parasite,  colors  the  iodophilic  inclusions.  Thin 
smears  may  be  stained  with  iron  hematoxylin  and  with  eosin  methylene 
blue  mixtures. 

The  flagellates  may  be  cultivated  on  media  used  for  the  cultural 
amoebae. 

GENUS  1.— Trichomonas  (Donne,  1837).— These  have  pyroform 
(pear-shaped)  bodies,  rounded  in  front  and  tapering  to  a  point 
behind,  provided  with  three  long  flagella,  often  matted  together  at 
the  anterior  end.  An  internal  supporting  structure,  known  as  the 
axial  filament  or  axostyle,  is  present.  There  is  an  undulating  mem- 
brane bordered  by  a  trailing  flagellum  that  begins  anteriorly  and 
runs  obliquely  backwards. 

Trichomonas  vaginalis  (Donne). — The  organism  is  fifteen  to 
twenty-five  microns  long  and  seven  to  twelve  wide;  it  is  provided 
with  three  flagella  and  an  undulating  membrane.  It  is  found  in 
the  vaginal  secretion  only  when  it  is  acid,  and  in  three  instances 
it  has  been  transmitted  to  the  male. 

Trichomonas  intestinalis  (R.  Leuckard,  1879).— This  parasite  is 
practically  indistinguishable  from  Trichomonas  vaginalis.  It  occurs 
in  the  small  intestine  and  appears  in  the  stools  during  diarrheal 
attacks,  but  is  probably  non-pathogenic.  It  is  readily  found  in  the 
intestine  and  colon  of  mice  and  guinea-pigs.  In  fresh  specimens 

1073 


1074 


PATHOGENIC   PROTOZOA 


(protected  with  a  cover  glass  and  vaseline)  it  is  actively  motile, 
but  the  undulating  membrane  is  difficult  to  detect  until  the  move- 
ment has  slowed  down. 

The  presence  of  cystic  forms  has  been  questioned,  and  two  quite 
different  forms  have  been  called  resistance  or  dauer  cysts.  The 

earlier  one,  described  by  Ucke  (1908), 
Bohne  and  von  Prowazek  (1908),  and 
Benson  (1910),  is  a  fairly  large  body, 
showing  a  double  contour  and  a  central 
homogeneous  mass,  perhaps  food  material, 
and  an  outer  ring-like  body  containing 
two  or  more  nuclei.  Brumpt  and  Alexieff 
believe  this  form  to  be  a  fungus,  having 
no  relation  to  the  trichomonad,  and  have 
called  it  ' '  Blast ocystis  hominis. ' '  Lynch1 
agrees  with  these  authors,  and  describes 
an  altogether  different  body  as  the  re- 
sistant form.  It  is  six  by  eight  microns 
in  size  and  perfectly  symmetrical  in 
shape.  The  wall  is  distinct,  and  there 
is  a  clear  space  between  it  and  the  body 
of  the  parasite.  The  nucleus,  undulating 

membrane  and  flagella  remain  visible  in 
FIG.    132\-TmcHOMONAS  IN-      the         t    but  L        h  was  unable  to  detect 
TESTINALIS.    (After  Brumpt,  .  . 

"Precis    de   Parasitologie,"      an*v  change  m  the  parasite  indicating  m- 
1914  ed.)  tracystic  multiplication. 

Infection  takes  place  probably  by  con- 
tact, and,  as  in  typhoid  fever,  food,  fingers  and  flies  carry  the  resistant 
forms  from  one  individual  to  another.  Among  the  natives  of  tropical 
countries  infection  is  almost  universal,  but  the  parasites  are  rarely 
seen  in  the  large  cities  of  the  North. 

GENUS  2. — Tetramitis  mesnili  (Wenyon,  1910). — Macrostoma  mes- 
nili,  Ckilomastix  mesniU,  Fanapapea  intestinalis.  This  organism,  first 
described  by  Wenyon,  from  a  native  of  the  Bahamas,  differs  from 
trichomonas  by  the  possession  of  a  deep  groove  or  cystostome,  in 
which  is  found  the  undulating  membrane.  It  is  present  in  diarrheal 
discharges,  but  its  pathogenicity  is  doubtful. 

GENUS  3. — Giardla  intestinalis  (synonym,  Lamblia  intestinalis). 
— The  giardia  are  bilaterally  symmetrical,  pear-shaped  organisms, 

1  Lynch,  Kenneth  M.,  Jour.  Parasitol.,  Urbana,  1916,  iii,  28. 


MASTIGOPHORA  1075 

provided  with  a  sucking  disk  anteriorly.  There  are  eight  pairs  of 
fiagella,  the  two  posterior  ones  being  continuations  of  longitudinal 
axostyles.  The  nucleus  is  first  dumb-bell  shaped  and  later  divided 
into  two  separate  nuclei.  Cysts  are  found,  and  according  to  Scliau- 
dinn  conjugation  occurs  in  them  with  the  development  of  four 
nuclei.  The  young  parasites  attach  themselves  to  the  surface  of 
epithelial  cells  of  the  small  intestine  by  the  sucking  disk,  but  even 
when  present  in  large  numbers  do  not  produce  any  characteristic 
symptoms.  Giardia  infestation  is  quite  common  in  children  in  the 
United  States,  and  not  uncommon  in  adults.  Maxcy*  found  20  per 


FIG.  133. — LAMBLIA  INTESTINALIS.     Cyst  formation.     (After  Doflein,  "  Lehrbuch  der 

Protozoenkunde . " ) 

cent  of  children  infested  and  Kofoid  found  6  per  cent  of  young 
healthy  soldiers  harboring  the  parasite.  The  same  or  like  parasites 
are  present  in  mice,  rats,  dogs,  cats,  and  sheep.  Transmission  is 
by  contact,  as  in  trichomonad  infections. 


ORDER   II— PROTOMONADINA 

The  Protomonadina,  another  order  of  the  Flagellata,  have  less  than 
three  flagella,  and  are  divided  into  the  Cercomonadidce,  Bodonidce  and 
the  Trypanosomidce. 

GENUS  1. — Cercomonadidae — Cercomonas  hominis  (Davaine, 
1854). — As  originally  described,  this  organism  has  a  pear-shaped  body, 

*  Maxcy,  Kenneth  F.,  Johns  Hopkins  Hospital  Bull.,  1921,  32,  166. 


1076  PATHOGENIC  PROTOZOA 

drawn  out  to  a  point  posteriorly,  is  armed  with  a  single  flagellum  in 
front,  and  has  no  undulating  membrane.  It  is  a  doubtful  species  and 
of  no  present  importance. 

GENUS  2. — Bodonidae — Prowazekia  (Hartmami  and  Chagas). — 
These  organisms,  the  only  examples  of  the  Bodonidce  of  medical  inter- 
est, are  of  some  importance,  since  they  have  been  cultivated  from 
human  feces  on  agar  plates.  The  genus  was  founded  for  Prowazekia 
cruzi,  a  species  discovered  in  human  feces  in  Brazil.  Other  species  are 
urinaria,  asiatica,  parva,  weinbergi  and  javanensis.  There  are  two 
flagella,  arranged  in  the  heteromastigote  manner,  that  is,  one  flagellum 
projects  forward  and  one  trails  behind.  There  is  no  undulating  mem- 
brane, but  in  stained  specimens  a  second  nucleus  is  seen,  the  kineto- 
nucleus  or  blepharoplast.  They  are  also  found  in  water,  and  are 
probably  not  the  cause  of  any  disease. 

GENUS  3. — Trypanosomidse. — History  of  the  genus. — In  1841  Val- 
entine discovered  the  first  hemoflagellate  in  the  blood  of  a  trout  now 
known  as  Trypanoplasma  valentini*  and  the  following  year  Gruby 
described  a  flagellate  in  frog's  blood  and  named  it  a  ' ' trypanosome. ' ' 
It  was  not  until  1878  that  Lewis  discovered  the  rat  trypanosome, 
Trypanosoma  lewisi.  The  first  pathogenic  member  of  the  genus  was 
noted  by  Evans  in  1889  in  the  blood  of  Indian  horses  sick  with  surra, 
Trypanosoma  evansi.  Bruce  in  1894  described  the  trypanosome  of 
Nagana,  Trypanosoma  Irucei  a  horse  disease  of  Zululand,  and  also 
demonstrated  its  transmission  by  the  tsetse  fly,  glossina  palpalis.  In 
1894  to  1899,  Rouget,  Schneider  and  Buffard  found  the  trypanosome 
of  Dourine,  or  "mal  de  coit,"  among  Algerian  horses.  Elmassian 
described,  in  1901,  the  South  American  horse  disease,  "mal  de 
caderas,"  and  discovered  the  parasite,  Trypanosoma  equinum.  Since 
this  time  a  large  number  of  new  species  have  been  discovered,  the 
more  important  of  which  will  be  described. 

Morphology. — The  morphology  of  the  trypanosomes,  while  subject 
to  many  variations  in  detail,  is  still  uniform  as  to  the  characteristics 
of  the  genus,  so  that  there  is  little  difficulty  in  immediately  recognizing 
the  parasite.  The  body  is  long  and  sinuous,  tapering  anteriorly  to  a 
fine  point  called  the  flagellum;  the  posterior  end  is  never  so  delicate 
and  is  often  quite  blunt.  All  contain  two  nuclei,  the  larger  being 
called  the  trophonucleus  and  the  smaller  the  kinetonucleus.  The 
trophonucleus  is  usually  located  midway  in  the  length  of  the  body, 

*  Gauthier,  M.  C.  E.  Acad.  Sci.,  1920,  170,  69. 


MASTIGOPHORA  1077 

and  the  kinetonucleus  behind  it,  often  at  the  posterior  extremity.  The 
flagellum  arises  from  a  centriole  (blepharoplast),  which  is  located 
close  to  or  in  the  kinetonucleus,  and  quickly  reaches  the  surface  of  the 
body,  when  it  turns  forward  and  forms  the  border  of  the  undulating 
membrane,  a  thin  fold  of  periblast  running  the  entire  length  of  the 
body,  and  is  often  continued  further  forward  as  delicate  filament. 
During  life  the  undulating  membrane  has  a  constant  wave-like  motion. 

Transmission  from  one  animal  to  another  is  usually  by  means  of 
some  blood-sucking  invertebrate.  Two  possible  forms  of  transmission 
have  been  recognized,  the  direct  and  indirect  or  cyclical;  the  direct 
form  is  used  in  the  laboratory  when  transferring  blood  with  a  hypo- 
dermic syringe  from  an  infected  animal  to  a  healthy  one,  and  it  also 
occurs  in  nature,  although  not  so  frequently  as  the  second.  Dourine, 
or  mal  de  coit,  is  the  best  example  of  the  natural  direct  method.  The 
cyclical  method  is  exemplified  in  the  transmission  of  Trypanosoma 
leAvisi  by  the  rat  flea,  Ceratophyllus  fasciatus,  in  which  insect  the 
trypanosome  passes  through  a  complicated  life  cycle.  Whether  the 
parasite  in  the  insect  ever  passes  from  parent  to  offspring  is  still 
doubtful.  Among  fishes,  reptiles  and  amphibians  the  parasites  are 
carried  by  leeches,  in  whose  intestinal  tract  they  undergo  a  cycle  of 
development. 

Just  as  in  malaria,  there  is  usually  an  alternation  of  hosts,  from 
invertebrate  to  vertebrate,  a  part  of  the  life  cycle  being  passed  in  each. 
In  the  blood  of  the  vertebrate  is  found  the  fully  developed  trypano- 
some, and  in  the  intestinal  tract  of  the  invertebrate,  crithidial.  and 
trypanomonad  types,  which  are  characterized  by  having  the  kineto- 
nucleus placed  in  front  of  or  close  beside  the  trophonucleus  and  by 
having  a  rudimentary  undulating  membrane. 

Cultivation. — In  1903  Novy  and  MacNeal2  first  obtained  pure  cul- 
tures of  trypanosomes  on  artificial  media.  The  medium  devised  by 
them  is  prepared  by  equal  parts  of  nutrient  agar  and  defibrinated  rab- 
bit blood.  After  the  agar  has  been  melted  and  cooled  to  about  50° 
C.  an  equal  quantity  of  rabbit  blood  is  added,  mixed  and  allowed 
to  cool.  "The  tubes  thus  prepared  are  allowed  to  set  in  an  inclined 
position,  after  which  they  are  at  once  inoculated.  It  is  essential 
that  the  surface  of  the  medium  be  moist  and  soft,  and  if  this  is 
not  the  case,  the  tubes  should  be  placed  in  an  upright  position  until 
some  water  of  condensation  accumulates  at  the  bottom.  The  initial 


2  Novy  and  MacNeal,  Contrib.  Med.  Eesearch    (Vaughan),  Ann  Arbor,   1903, 
p.  549. 


1078  PATHOGENIC   PROTOZOA 

culture  usually  requires  a  week  or  more,  although  not  infrequently 
fairly  rich  growths  may  be  obtained  in  three  or  four  days"  (Novy). 

Trypanosoma  rotatorium. — Gruby  described  and  named  this 
hemoflagellate  in  1843,  and  it  is,  therefore,  the  type  species  of  the 
genus.  The  organism  is  widely  distributed  throughout  the  world, 
and  is  found  in  Rana  esculenta,  Rana  temporaria  and  Hyla  arborea: 
the  organisms  are,  however,  not  very  numerous  in  any  single  frog. 
It  is  most  often  found  during  the  spring  and  summer  months,  rarely 
in  winter. 

Morphology. — Both  body  and  undulating  membrane  are  broad, 
the  cytoplasm  is  granular,  and  toward  the  straight  side  shows  striae, 
probably  indicating  the  presence  of  myonemes.  The  trophonucleus 


FIG.  134.— TRYPANOSOMA   ROTATORIUM    IN    BLOOD    OF    FROG.     (After    MacNeal 
"Pathogenic  Microorganisms,"  published  by  P.  Blakiston's  Son  &  Co.) 

(s  large,  lies  near  the  middle,  of  the  body  and  near  the  undulating 
membrane;  the  kinetonucleus  is  smaller,  lies  posteriorly  and  stains 
deeply;  the  flagellum  which  originates  near  the  kinetonucleus  turns 
forward,  forming  the  border  of  the  undulating  membrane,  and  is 
continued  forward  as  a  short  flagellum.  The  posterior  end  is  usually 
drawn  out  to  a  stubby  point.  The  fully  developed  organism  is  large, 
being  40  to  80  microns  long  by  5  to  40  wide.  One  striking  thing 
about  this  parasite  is  its  tendency  to  pleomorphism. 

Multiplication  in  the  blood  stream  of  the  frog  is  by  binary  fission ; 
in  addition,  a  form  of  multiple  division  occurs  in  the  viscera,  pre- 
ceded, according  to  Machado,  by  conjugation  of  sexually  differen- 
tiated forms.  The  merozoites  liberated  from  the  mother  cell  are 
small  trypanosomes,  which  in  turn  grow  to  large  size,  thus  explaining 
the  pleomorphism  of  the  parasite. 


MASTIGOPHORA 


1079 


Cultures  have  been  obtained  by  Lewis  and  Williams  on  the  blood 
a  gar  of  Novy  and  MacNeal  in  which  a  great  variety  of  forms  may 
be  seen ;  the  method  of  transmission  is  imkown,  but  the  infection  is 
probably  conveyed  by  leeches.  Many  other  trypanosomes  have  been 
found  in  fishes,  frogs,  and  reptiles  all  over  the  world. 

Trypanosoma  lewisi  (Kent). — This,  one  of  the  longest  known  and 
commonest  forms,  has  been  studied  more  completely  than  any  other 
organism  of  its  class.  It  occurs  in 
a  large  proportion  of  rats  through- 
out the  world,  twenty-five  to  one 
hundred  per  cent  being  infected, 
and  since  it  is  non-pathogenic,  it  is 
a  convenient  organism  for  research. 
It  may  be  passed  fr^m  wild  to 
white  rats  without  difficulty,  by  in- 
oculating the  latter  with  a  small 
quantity  of  citrated  blood  contain- 
ing the  organisms.  At  first  the  para- 
sites are  few,  but  after  the  lapse 
of  three  or  four  days,  lafcge  numbers 
may  be  found;  the  condition  of 
rapid  multiplication  lasts  from  eight 
to  fourteen  days,  and  is  succeeded 
by  a  period  of  a  month  or  more, 
during  which  time  the  parasites 
gradually  diminish  in  number,  finally 
disappearing  completely,  rendering 
the  animal  immune  from  further  infection,  the  immunity  being  com- 
plete. The  serum  of  an  immune  rat  has  a  certain  protective  power, 
and  when  inoculated  simultaneously  with  blood  containing  trypano- 
somes, may  prevent  the  infection.  No  other  animals  are  susceptible. 

The  blood  should  be  examined  in  both  fresh  and  stained  speci- 
mens. In  fresh  specimens,  because  of  the  rapid,  lashing  movements 
of  the  parasite,  the  organisms  are  particularly  easy  to  find.  The 
details  of  structure,  however,  do  not  appear  except  in  spreads 
stained  with  some  of  the  modifications  of  the  Romanowski  stain, 
such  as  Wright's  or  MacNeal 's. 

In  the  adult  stage  the  organisms  are  quite  uniform  in  size  and 
shape,  being  27  or  28  microns  long  and  1.5  to  2.0  microns  broad; 
the  posterior  end  is  long,  tapering  and  pointed;  the  kinetonucleus 


FIG.  135. — TRYPANOSOMA  LEWISI. 
(After  Doflein  and  Minchin.  Mac- 
Neal, "  Pathogenic  Microorgan- 
isms," published  by  P.  Blakiston's 
Son  &  Co.) 


1080  PATHOGENIC  PROTOZOA 

oval  and  flattened;  the  trophonucleus  is  located  near  the  anterior 
end,  and  the  undulating  membrane,  while  distinct,  is  relatively 
narrow.  The  endoplasm  is  finely  granular,  and  by  careful  focusing 
the  bodywall  or  periblast  may  be  seen. 

Multiplication  in  the  rat  is  rapid,  and  many  young  forms  are 
seen;  these  are  smaller,  stain  more  deeply,  and  vary  much  more  in 
size  than  the  adults.  Dividing  forms  are  common,  the  division  being 
longitudinal  and  unequal,  the  parent  retaining  the  flagellum.  Mul- 
tiple division  also  occurs,  resulting  in  the  production  of  rosettes, 
whose  structure  suggests  that  repeated  longitudinal  division  has 
occurred  without  the  separation  of  the  daughter  cells. 

The  insect  hosts  are  two :  the  rat  flea,  Ceratopliyllus  fasciatus,  and 
the  rat  louse,  Hcematopinus  spinulosus;  the  former  being  the  right 
host  and  the  latter  the  wrong  one,  since  in  it  development  is  incom- 
plete. Minchin  and  Thompson3  have  studied  the  cycle  in  the  flea, 
which  is  briefly  as  follows:  When  the  injected  blood  and  parasites 
reach  the  midgut  of  the  flea,  the  trypanosomes  lose  their  flexibility 
and  become  more  or  less  rigid,  and  are  able  to  penetrate  the  outer 
wall  of  the  epithelial  cells  of  the  stomach.  Once  inside  the  cell, 
the  parasite  folds  upon  itself  and  grows  to  large  size;  the  nuclei 
multiply,  the  body  becomes  spherical  and  divides  up  within  its  own 
periblast  into  six  or  eight  daughter  cells,  all  actively  moving  within 
their  common  envelope.  This  becomes  tense  and  finally  bursts, 
liberating  the  young  trypanosomes  within  the  epithelial  cell,  through 
whose  wall  they  soon  escape  into  the  lumen  of  the  stomach.  This 
form  of  multiplication  may  be  several  times  repeated,  after  which 
the  young  trypanosomes  pass  down  the  intestine  to  the  lower  end 
to  begin  the  rectal  phase.  There  the  parasites  in  large  numbers  are 
found  attached  to  the  epithelial  cells  by  their  flagella.  Rapid  mul- 
tiplication takes  place  by  repeated  fission  and  the  parasite  becomes 
crithidial  in  form,  that  is,  it  loses  its  undulating  membrane,  becomes 
short  .and  stubby,  and  the  kinetonucleus  moves  forward  close  to  or 
in  front  of  the  trophonucleus.  Ultimately  some  change  back  to 
minute  trypanosomes,'  and  these,  when  regurgitated  or  passed  in 
the  feces,  serve  to  infect  the  next  victim.  The  rectal  phase,  when 
once  established,  lasts  for  several  months  or  perhaps  indefinitely, 
making  every  infected  flea  a  chronic  carrier. 

Trypanosoma  evansi. — Surra  is  a  disease  of  horses  and  mules, 
camels,  elephants,  buffaloes,  and  dogs,  which  prevails  in  India  and 

8  Minchin  and  Thompson,  Quarter.  Jour.  Micr.  Sc.,  Lond.,  1915,  Ix,  463. 


MASTIGOPHORA  108 1 

other  parts  of  Asia,  and  also  in  the  Philippines  and  Northern  Aus- 
tralia. The  Philippine  outbreak  was  traced  to  animals  returned 
from  China  after  the  Boxer  outbreak;  for  at  that  time  American 
troops  came  into  contact  with  native  Indian  troops  and  their 
animals. 

The  trypanosome  causing  the  disease  was  discovered  by  Evans 
in  1880.  The  clinical  course  of  the  disease  is  marked  by  an  irregular 
recurring  fever,  with  many  remissions,  during  which  the  parasite 
cannot  be  demonstrated  in  the  blood,  although  it  is  not  difficult  to 
find  during  the  febrile  period.  The  animal  is  anemic,  weak, 
emaciated,  and  may  show  an  ecchymotic  eruption  on  the  abdomen. 
The  course  of  the  disease  may  be  either  short  or  long,  but  leads 
almost  invariably  to  death.  In  camels  it  lasts  from  two  to  four  years, 
often  without  symptoms  until  near  the  end,  and  these  animals  prob- 
ably act  as  chronic  carriers. 

Morphology. — Morphologically,  the  parasite  is  very  like  the  Tryp- 
anosoma  brucei  of  Nagana,  yet,  as  a  rule,  the  trophonucleus  lies  nearer 
the  anterior  end  than  in  brucei,  although  it  may  be  impossible  to 
distinguish  in  smears  between  the  two. 

The  disease  is  carried  by  biting  flies,  tabanidoe,  and  stomoxys,  and 
also  by  fleas. 

Trypanosoma  brucei. — Nagana  is  a  well-known  horse  and  animal 
disease  of  Africa,  which  causes  an  enormous  economic  loss  and  has 
greatly  interfered  with  the  development  of  the  country.  The  parasite 
was  discovered  by  Bruce  in  1895.  Among  the  natives  it  is  known 
as  tsetse  fly  disease,  and  investigation  has  incriminated  Glossina  mor- 
sitans  as  the  carrier.  Clinically,  the  disease  in  horses  is  much  like  the 
Surra  of  India ;  the  native  name  for  the  disease,  nagana,  means  weak- 
ness. Nearly  all  the  larger  animals  are  susceptible  to  either  natural  or 
artificial  infection,  yet  man  is  apparently  immune., 

Morphology. — Morphologically,  it  resembles  closely  most  of  the 
other  pathogenic  trypanosomes,  and  Minchin  makes  it  the  type  of  a 
group  of  pathogenic  trypanosomes,  all  closely  resembling  one  another 
and  possibly  descended  from  one  common  ancestor :  the  group  consists 
of  brucei,  gambiense,  evansi,  equiperdum,  rhodesiense,  and  hippicum. 
The  organism  is  less  slender  than  Icwin  and  has  a  wider  undulating 
membrane.  The  posterior  end  is  relatively  short,  the  trophonucleus 
lies  in  the  middle  of  the  body  and  the  kinetonucleus  at  the  extreme 
posterior  end ;  a  vacuole  is  placed  just  in  front  of  the  latter.  In  length 
the  parasite  measures  twenty-five  to  thirty-five  microns  and  is  one  and 


1082  PATHOGENIC  PROTOZOA 

a  half  to  two  and  a  half  microns  in  width ;  multiplication  in  the  blood 
stream  is  by  binary  fission. 

Transmission  is  by  means  of  the  tsetse  fly,  Glossina  morsitans,  and 
perhaps  Glossina  pallidipes  and  Glossina  fusca.  The  fly  may  transmit 
the  disease  directly  after  infection,  acting  as  a  mere  mechanical 
carrier,  but  it  is  more  probable  that  a  cyclical  development  of  the 
parasite  takes  place  in  the  fly,  after  which  it  remains  infectious  for 


D 


FIG.  136. — THE  MOST  IMPORTANT  TRYPANOSOMES  PARASITIC  IN  VERTEBRATES. 
A,  Tr.  lewisi;  B,  Tr.  evansi  (India);  C,  Tr.  evansi  (Mauritius);  D,  Tr.  brucci; 
E,  Tr.  equiperdum;  F,  Tr.  equinum;  G,  Tr.  dimorphon;  H,  Tr.  gambiense. 
(X1500.)  (From  Doflein  after  Novy.  MacNeal,  "  Pathogenic  Microor- 
ganisms," published  by  P.  Blakiston's  Son  &  Co.) 

a  long  period.  It  has  been  shown  that  after  the  first  few  hours 
the  fly  is  not  infectious  again  until  the  lapse  of  eighteen  days,  when 
its  bite  once  more  conveys  the  disease,  and  trypanosomes  may  be 
found  in  the  intestinal  canal,  the  body  cavity,  the  salivary  glands 
and  in  the  proboscis.  Studies  of  the  cycle  in  the  fly  show  that 
only  about  five  per  cent  of  the  flies  permitted  to  feed  on  sick  animals 
become  chronic  carriers.  The  work  of  Bruce  and  others  has  shown 
that  the  trypanosomes  are  more  or  less  harmless  parasites  of  the 
big  game  animals  of  Africa,  which  therefore  are  believed  to  act 
as  a  reservoir,  from  which  the  disease  is  transferred  to  the  domestic 


MASTIGOPHORA  1083 

animals  by  the  tsetse  fly.  The  distribution  of  Glossina  is  not  uni- 
form, as  they  are  only  present  in  certain  definite  areas  called  fly 
belts.  Since  the  disease  does  not  spread  in  the  absence  of  the 
larger  wild  animals,  it  has  been  proposed  that  all  big  game  be 
exterminated  as  a  prophylactic  measure.  Mice  and  rats  are  sus- 
ceptible and  die  in  six  to  fourteen  days  after  inoculation;  guinea- 
pigs  are  more  resistant,  and  may  show  one  or  more  relapses  within 
two  to  ten  weeks.  It  has  not  been  possible  to  immunize  larger 
animal's,  although  a  certain  degree  of  success  has  been  obtained 
with  the  smaller  animals  used  in  the  laboratory. 

Cultures  have  been  grown  on  artificial  media,  yet  not  so  readily 
as  with  lewisi  and  avian  trypanosomes.  The  medium  recommended 
by  MacNeal  contains  the  extractives  of  one  hundred  and  twenty-five 
grams  of  meat,  ten  of  pepton,  five  of  salt,  and  twenty-five  of  agar 
to  the  liter ;  to  this  is  added  twice  its  volume  of  warm,  defibrinated 
rabbit 's  blood.  The  blood  agar  slants  should  be  soft  and  moist  when 
inoculated.  Filtrates  from  cultures  are  not  toxic,  the  toxin  ap- 
parently being  liberated,  according  to  MacNeal,  from  the  body  of 
the  disintegrating  trypanosome. 

Trypanosoma  hippicum  (Darling). — The  disease  caused  by  this 
trypanosome  in  horses  and  mules  has  been  known  in  Panama  for 
many  years  under  the  name  of  "Murrina  de  caderas"  or  "Der- 
rengadera  de  caderas,"  the  latter  term  being  used  when  paralysis 
of  the  posterior  extremities  is  the  dominant  symptom;  both  names 
indicate  a  weakness  of  the  hind  quarters.  The  symptoms  are  weak- 
ness, emaciation,  and,  sooner  or  later,  conjunctivitis  and  subcon- 
junctival  ecchymosis,  and  anemia.  The  horses  and  mules  affected 
are  obviously  weak,  and  while  in  the  stall,  pull  back  on  the  halter, 
or  stand  with  straddling  hind  legs. 

The  incubation  period  in  animals  used  for  experiment  is  less 
than  a  week,  a  few  animals  lose  weight  rapidly  and  die  within  a  few 
days,  others  live  for  several  weeks. 

Treatment,  including  the  use  of  arsenical  preparations,  is  without 
effect,  and  all  infected  animals  should  be  destroyed. 

The  disease  is  apparently  transmitted  directly  by  flies,  which 
carry  blood  and  serum  from  ulcers  and  abrasions  on  infected  to 
healthy  animals. 

Morphology  of  the  Parasite. — The  trypanosome  is  sixteen  to 
eighteen  microns  long  and  two  microns  wide.  The  kinetonucleus 
is  about  two  microns  from  the  posterior  end,  the  trophonucleus 


1084  PATHOGENIC  PROTOZOA 

about  eight  to  ten  microns  from  the  same  point.  The  posterior  end 
is  blunt  and  the  cytoplasm  usually  contains  numerous  basophile 
granules;  the  undulating  membrane  is  well  developed  and  a 
chromatin  filament  runs  from  the  kinetonucleus  to  the  tip  of  the 
flagellum.  The  large  kinetonucleus  distinguishes  this  organism  from 
Trypanosoma  equinum. 

PatJwlogical  Anatomy. — Aside  from  the  emaciation,  edema  of  the 
belly  wall,  conjunctivitis  and  subconjunctival  ecchymosis,  there  is 
usually  excessive  fluid  in  the  body  cavities,  an  enlarged  spleen,  and, 
what  is  more  characteristic,  small  petechial  spots  on  the  capsule 
of  the  spleen  and  in  the  cortex  of  the  kidney  and  in  the  endo-  and 
pericardium  and  occasionally  on  the  pleura!  surfaces. 

Prophylaxis  consists  in  the  destruction  of  all  infected  animals; 
the  protection  of  wounds  and  ulcers  in  otherwise  healthy  animals 
by  dressings  and,  wherever  possible,  the  use  of  fly  screens  about 
the  stables. 

Trypanosoma  equiperdum  (Do urine). — This  organism  is  the 
cause  of  dourine,  a  disease  of  horses  and  donkeys,  which  is  usually 
transmitted  by  coitus,  but  may  be  carried  by  biting  flies,  stomoxys. 
The  organism  was  first  described  by  Rouget  in  1894;  it  resembles 
brucei  in  many  ways  and  produces  a  progressive,  fatal  disease  of 
great  economic  importance.  Formerly  it  was  present  throughout 
the  greater  part  of  Europe,  but  is  now  almost  limited  to  the  shores 
of  the  Mediterranean.  From  time  to  time  it  has  been  introduced 
into  the  United  States  and  Canada  by  blooded  French  stallions  and 
has  spread  into  parts  of  the  Northwest. 

The  clinical  course  may  be  divided  into  a  stage  of  edema,  lasting 
about  a  month,  during  which  there  is  a  painless,  soft  swelling, 
limited  to  the  genitalia  and  the  belly  wall.  This  is  followed  by 
the  stage  of  eruption,  during  which  plaques,  or  round  edematous 
areas,  are  found  under  the  hide  on  the  flanks  and  hind  quarters, 
and  sometimes  on  thighs,  shoulders  and  neck;  this  stage  is  short, 
lasting  about  a  week.  It  is  followed  by  the  third  stage  of  paralysis 
and  anemia ;  the  animal  loses  flesh  and  strength,  develops  superficial 
ulcers,  conjunctivitis,  keratitis,  and  ultimately  paralysis,  leading  to 
death  in  two  to  eighteen  months. 

The  trypanosome  is  found  most  readily  in  the  serous  exudate 
from  the  ulcers,  as  it  is  infrequent  in  the  peripheral  circulation; 
in  this  respect  it  resembles  the  treponema  of  lues.  The  organism 
is  about  twenty-five  microns  in  length  and  possesses  a  clear  cyto- 


MASTIGOPHORA 


1085 


plasm,  free  from  granules,  except  when  propagated  in  white  mice, 
when  they  are  plentiful. 

Diagnosis  by  Complement  Fixation. — E.  A.  Watson,4  of  Canada, 
has  shown  that  it  is  possible  not  only  to  diagnose  the  disease  when 
the  clinical  signs  are  clear,  but  also  to  determine  the  existence  of 
its  non-clinical,  obscure  and  latent  forms.  Horses  may  tolerate  an 
infection  for  one  to  three  years,  during  which  time  they  arc  capable 
of  conveying  the  disease  and  yet  remain  normal  in  health  and 


FIG.  137. — DOURINE.  Showing  swelling  of  genitalia  and  plaques  on  the  skin. 
(After  Kolle  and  Wassermann,  "Handbuch  der  Pathogenen  Mikro-organismen," 
2te  Aufl.,  1913.) 

general   appearance,   and   this   method  of   diagnosis   is,    therefore, 
invaluable. 

Watson  obtains  the  antigen  by  inoculating  a  large  number  of 
white  rats  with  Trypanosomv,  equiperdum,  collecting  their  blood  when 
teeming  with  trypanosomes,  and  separating  them  from  the  erythro- 
cytes  and  plasma  by  washing  and  centrifuging.  Each  of  ten  to 
twenty  rats  receive  0.3  c.c.  of  blood  rich  in  trypanosomes  intraperi- 
toneally,  and  at  about  the  end  of  the  third  day,  when  the  organisms 
are  very  numerous,  the  rats  are  bled  into  citrate  solution.  By 


.  A.  Watson,  Parasitology,  Cambridge,  Eng.,  1915,  VIII,  156. 


1086 


PATHOGENIC   PROTOZOA 


repeated  washing  the  organisms  may  be  separated,  as  a  pure  white 
layer  overlying  the  erythrocytes.  This  mass  of  organisms  is  killed 
and  preserved  by  a  formalin-glycerin  mixture,  after  which  its  an- 
tigenic  strength  is  standardized  by  titration  in  the  usual  way.  The 
test,  a  pure  culture  of  trypanosomes  being  used  as  antigen,  is  specific 
and  is  not  positive  in  any  .other  disease  of  horses. 

Trypanosoma  avium. — This  parasite  was  first  described  by  Dani- 
lewski  in  1885.  In  1905  .Novy  and  .MacNeal5  .found  trypanosomes 
in  8.8  per  cent  of  431  American  birds.  Although  there  are  doubt- 
less several  species,  the  most  common  is  Trypanosoma  avium,  a  para- 


FIG.  138.— TRYPANOSOMA  AVIUM  IN  BLOOD  OF  COMMON  WILD  BIRDS.  (After  Novy 
and  MacNeal.  MacNeal,  " Pathogenic  Microorganisms,"  published  by  P. 
Blakiston's  Son  &  Co.) 

site  twenty  to  seventy  microns  long  and  four  to  seven  microns  wide. 
They  are  found  in  the  blood  over  long  periods  of  time  and  do  not 
appear  to  be  pathogenic.  Cultures  are  easily  made  and  kept  alive 
for  long  periods  by  weekly  transfers.  The  mode  of  transmission  is 
unknown. 

This  was  the  parasite  which  was  confounded  in  1904  by  Schaudinn 
with  developmental  stages  in  the  life  cycle  of  Hemorproteus  noctuce 
and  Hemorproteus  ziemani  with  resulting  confusion  in  the  study  of 
trypanosomes  and  hemocytozoa,  and  it  is  only  recently  that  the  error 
has  been  generally  acknowledged. 

Trypanosoma  gambiense  (Sleeping  Sickness). — Two  names  have 
been  given  to  the  disease  caused  by  this  parasite,  both  of  which  are 


9  Novy  and  MacNeal,  Jour,  Infect.  Dis.,  Chicago,  1905,  ii,  256. 


MASTIGOPHORA 


1087 


now  recognized  as  stages  in  one  and  the  same  infection,  human  tryp- 
anosomiasis :  they  were  trypanosome  fever,  and  sleeping  sickness.  It 
is  a  chronic  infection  characterized  by  fever,  lassitude,  weakness, 
wasting,  and,  in  its  terminal  stages,  by  a  protracted  lethargy. 
Sleeping  sickness  and  trypanosome  fever  had  long  been  known  in 
tropical  Africa,  and  the  disease  at  present  is  widespread  and  the 


FIG.  139. — TRYPANOSOMA  AVIUM  IN  CULTURE  ON  BLOOD  AGAR.  (X1500).  (After 
Novy  and  MacNeal.  MacNeal,  " Pathogenic  Microorganisms,"  published  by 
P.  Blakiston's  Son  &  Co.) 

cause  of  tremendous  mortality.  It  is  estimated  that  one  hundred 
thousand  deaths  occurred  during  the  ten  years  ending  in  1910.  It 
is  endemic  in  the  lake  region  of  Central  Africa,  and  in  the  Congo 
basin.  It  was  early  introduced  into  Martinique  in  the  West  Indies, 
but  did  not  spread  and  has  now  died  out. 

Button  and  Todd  found  the  parasite  in  1901  in  the  blood  of  an 
Englishman  in  Gambia,  who  died  after  a  febrile  illness  of  two  years' 


1088 


PATHOGENTC  PROTOZOA 


duration ;  Castellan!  in  1903  found  the  parasite  in  the  cerebro-spinal 
fluid  of  well-marked  eases  of  sleeping  sickness  occurring  among 
natives  of  Uganda. 

Clinical  Signs. — The  disease  begins  with  slight  febrile  attacks, 
headache  and  increasing  weakness,  emaciation,  swelling  of  the  eyelids 
and  enlargement  of  the  lymph  nodes.  The  temperature  increases, 
edema  of  the  extremities  appears  and  the  spleen  enlarges.  During 
the  last  stages  nervous  symptoms  predominate  and  the  patient  sleeps 
day  and  night,  but  may  have  periods  of  excitement  or  convulsions, 
yet  finally  sinks  into  deep  coma  and  dies  of  exhaustion. 


FIG.  140. — TRYPANOSOMA  GAMBIENSE.     Calkin,  "Protozoology." 

Etiology. — The  disease  is  transmitted  by  the  bite  of  the  tsetse  fly, 
Glossina  palpalis,  which  is  apparently  able  to  transmit  the  infection 
mechanically  immediately  after  biting  an  infected  host,  yet  in  most 
flies  the  trypanosomes  disintegrate  and  disappear  from  the  intestinal 
tract  within  four  or  five  days.  In  from  five  to  ten  per  cent  of  the 
flies,  however,  the  trypanosomes  multiply  in  the  intestinal  tract,  and 
after  eighteen  to  fifty-three  days  they  again  become  infectious  and 
remain  so  for  a  long  period,  the  parasites  being  found  regularly  in 
the  salivary  glands  and  in  the  proboscis. 

It  is  possible  that  the  disease  is  transmitted  in  other  ways  than 
by  Glossina  palpalis;  blood-sucking  insects,  such  as  stomoxys, 


MAST1GOPHORA  -  1089 

anopheles,  mansonia  and  perhaps  fleas,  may  act  as  mechanical  carriers. 
It  is  also  possible  that  the  disease  is  transmitted  by  coitus.  Without 
some  such  explanation  it  is  difficult  to  understand  certain  house 
epidemics  which  have  occurred  outside  the  fly  belts. 

The  animal  host  of  the  Trypanosoma  gambiense  is  believed  to  be 
the  big  game  animals,  particularly  the  antelope. 


FIG.    141. — TSETSE    FLY    (GLOSSINA    PALPALIS).     (From    Rosenau,    "  Preventive 

Medicine  and  Hygiene.") 

Morphology. — The  organism  belongs  to  the  brucei  group,  and  its 
differentiation  on  morphology  is  difficult,  yet,  on  the  average,  the 
posterior  end  is  somewhat  more  pointed  than  the  brucei.  In  length 
it  varies  from  fifteen  to  thirty  microns,  and  in  thickness  from  one  to 
three  microns.  In  fresh  preparations  the  motility  is  not  marked; 
both  plump  and  slender  forms  are  found  in  the  blood,  but  in  the  cere- 
bro-spinal  fluid  slender  forms  only  are  seen. 

Cultures  on  blood  agar  have  been  made  by  Thompson  and  Sinton, 
yet  they  died  out  after  a  few  weeks,  and  were  never  virulent.  The 
pathogenicity  varies  somewhat  with  the  strain  used,  but  apes  are  easily 
infected.  In  white  rats  there  may  be  two  or  three  relapses  before 
death  occurs,  while  when  inoculated  with  brucei  death  follows  within 
two  weeks. 

Pathogenicity. — Although  cultures  vary  greatly  in  virulence,  it  is 
possible  to  infect  rats,  dogs  arid  monkeys  with  a  fatal  trypanosomiasis ; 
cattle,  sheep  and  goats  continue  to  show  a  few  parasites  for  months 


!090  PATHOGENIC  PROTOZOA 

after  inoculation  but  without  sickening.  In  no  animal,  however,  is 
it  possible  to  reproduce  the  sleeping  sickness  stage  as  it  occurs  in 
man. 

Trypanosoma  rhodesiense. — This  species  was  established  by 
Stephens  and  Fantham.6  It  is  transmitted  by  the  Glossina  morsitans, 
a  fly  which  is  widespread  over  large  tracts  of  country,  independently 
of  the  presence  of  water.  It  is  becoming  generally  recognized  that 
there  are  two  forms  of  sleeping  sickness,  one  of  which  is  caused  by 
this  trypanosome.  This  form  of  the  disease  is  more  acute  and  is 
less  amenable  to  treatment;  the  trypanosome  is  also  more  virulent 
for  animals  and  may  be  differentiated  from  gambiense  on  its 
morphology.  As  both  parasites  are  found  in  the  antelope,  the  prophy- 
laxis is  the  same.  Bruce7  is  of  the  opinion  that  rhodesiense  and 
brucci  are  identical,  but  Taute  and  Huber,*  by  inoculating  themselves 
and  129  natives  with  the  blood  of  naturally  infected  animals  with 
out  reproducing  the  disease,  seem  to  have  shown  that  the  parasites 
are  not  identical. 

Diagnosis. — When  the  disease  is  well  developed  in  an  endemic 
area,  the  diagnosis  is  easily  made.  During  the  early  stages  the  exami- 
nation of  the  cerebro-spinal  fluid,  puncture  fluid  from  the  lymph  nodes 
and  the  peripheral  blood  may  all  show  the  trypanosome;  since  the 
parasites  are  scarce  the  use  of  the  thick  film  method  of  Ross  may  be 
necessary.  When  direct  examination  is  unsuccessful,  enrichment  in 
the  blood  of  susceptible  animals,  rats  and  mice  will  establish  the 
diagnosis. 

Treatment. — Treatment  is  based  upon  the  observation  of  Bruce 
and  Lingard,  that  arsenious  acid  is  trypanocidal.  The  best  results 
have  been  obtained  with  atoxyl,  in  half  gram  doses,  repeated  at  inter- 
vals of  ten  days  or  more  for  not  less  than  four  months.  Light  cases 
become  trypanosome  free  and  are  apparently  cured,  yet  many  relapse 
on  cessation  of  treatment.  Well  marked  cases  may  show  improvement 
yet  ultimately  grow  worse  and  die.  Better  results  are  obtained  when 
atoxyl  and  tartar  emetic  are  both  used  and  also  when  the  treatments 
are  repeated  every  six  months.  The  successful  treatment  of  an  intract- 
able case  of  gambiense  infection  with  stibenyl  (the  sodium  salt  of 
p-acetylaminophenyl-stibinic-acid)  has  been  reported  by  Manson-Bahr 


'Stephens  and  Fantham,  Proc.  Eoy.  Soc.,  1910,  Ser.  B.,  Ixxxiii,  28. 

1  Bruce,  Bull.  Trop.  Dis.,  1916,  vii,  68. 

*  Taute,  M.,  and  Uulxr,  /•'.,  Abstracted  in.  Trouieal  Diseases.  Bull.  London,  1930. 


MAST1GOPHORA  1091 

•(Brit.  Med.  Jl.,  1920,  235).  Other  arsenical  preparations  have  been 
used  but  none  are  entirely  successful.  Salvarsan  drives  the  parasite 
from  the  peripheral  blood  but  not  from  the  cerebro-spinal  fluid.  The 
prognosis  as  to  ultimate  recovery  is  unfavorable.  The  most  hopeful 
cases  are  those  which  are  brought  under  treatment  in  the  earliest 
stage  of  the  disease. 

Prophylaxis. — Prophylaxis  is  quite  complicated  and  is  carried  out 
along  several  different  lines.  Infected  fly  belts  are  depopulated,  the 
inhabitants  being  removed  to  a  fly-free  district  where  they  may  be 
treated  at  hospital  stations.  The  fly  breeding  may  be  greatly 
diminished  by  clearing  off  the  forest  and  brush,  especially  along  the 
river  courses,  since  the  glossina  needs  abundant  moisture  for  its  propa- 
gation. Since  the  fly  bites  only  during  the  day,  all  traveling  in 
infected  districts  is  best  done  at  night.  A  prophylactic  measure  of 
prime  importance  is  the  search  for  and  the  treatment  of  the  cases, 
which  are  also  carriers.  While  the  treatment  may  not  cure,  it  does 
bring  about  improvement  and  lessens  the  number  of  heavily  infected 
carriers. 

Trypanosome  cruzi,  Chagas  (Schizotrypanum  cruzi). — This  para- 
site, which  differs  from  all  other  trypanosomes,  is  the  cause  of  a 


FIG.  142.  —  SCHIZOTRYPANUM  CRUZI  IN  HUMAN  BLOOD.  (From  Doflein  after 
Chagas.  MacNeal,  "Pathogenic  Microorganisms,"  published  by  P.  Blakis- 
ton's  Son  &  Co.) 

form  of  human  trypanosomiasis  occurring  in  Brazil.  It  is  trans- 
mitted by  a  bug,  Triatoma  magista  (Conorkinus  megistus),  in  which 
the  parasite  passes  part  of  its  cycle  of  development.  Brumptf  believes 
the  infection  is  transmitted  by  the  dejecta  of  the  bug  as  well  as  by 
its  bite,  and  thinks  the  first  method  is  probably  the  more  usual 
one.  In  the  human  being,  multiplication  takes  place  in  endothelial 
cells,  lymphocytes  and  parenchymatous  cells  of  the  viscera ;  and  also 
in  the  skeletal  and  heart  muscles.  While  in  this  stage  the  parasite 

|  Brumpt,  Bull,  Acad.  Med.,  Paris,  1919,  81,  251. 


1092 


PATHOGENIC  PROTOZOA 


FlG.  143. — SCHIZOTRYPANUM     CRUZI    DEVELOPING    IN    TISSUES    OF    GlJINEA-PIG.      1. 

Cross-section  of  fibers  of  striated  muscle  containing  Schizotrypanum  cruzi;  2, 
Section  of  brain  showing  cyst  in  a  neuroglia  cell  containing  chiefly  flagellated 
forms;  3,  Section  through  suprarenal,  fascicular  zone;  4,  Section  of  brain 
showing  neuroglia  cell  filled  with  round  forms.  (After  Low  and  Vianna. 
MacNeal,  "Pathogenic  Microorganisms,"  published  by  Blakiston's  Son  &  Co.) 


MASTIGOPHORA  1093 

has  no  flagellum  and  resembles  the  leishmania;  only  after  escape  into 
the  blood  does  it  take  011  the  trypanosome  form. 

Guinea-pigs,  rats,  mice  and  monkeys  are  susceptible;  the  bed- 
bug, cimex,  is  also  capable  of  transmitting  the  disease. 

Cultures  were  obtained  by  Chagas  and  proved  virulent  for 
animals.  The  human  disease  is  found  both  in  children  and  adults 
and  is  regularly  fatal.  It  is  characterized  by  an  irregular  fever, 
severe  anemia,  swelling  of  the  lymph  nodes,  edema  and  disturbance 
of  the  nervous  system.  Two  quite  distinct  forms  are  recognized, 
the  acute,  which  is  usually  found  in  young  children,  and  the  chronic, 
found  in  adults.  The  acute  form  is  characterized  by  fever,  a  myxe- 
dematous  swelling  of  the  face  and  neck  or  even  the  whole  body, 
and  the  presence  of  trypanosomes  in  the  circulating  blood.  The 
nervous  system  may  be  involved  in  the  acute  cases,  in  which  event 
they  end  fatally.  The  chronic  form  has  no  trypanosomes  in  the 
blood,  but  has  foci,  or  cysts  in  the  heart,  the  voluntary  muscles  or 
in  the  viscera  or  the  nervous  system.  Disturbances  of  almost  any 
part  of  the  body  may  result.  While  the  foci  may  be  generalized 
they  do  not  follow  the  blood  vessels. 

Leishmania. — This  genus  was  founded  by  Boss  in  1903  for  the 
Leishman-Donovan  and  Wright  bodies  found  in  kala-azar  and  Delhi 
boil,  to  which  Nicolle  added  another  in  1909,  the  parasite  of  infantile 
splenomegaly.  Leishman,  Donovan  and  Wright,  working  independ- 
ently, described  the  first  two  parasites  in  1903,  and,  although  they 
have  received  various  names,  leishmania  is  now  the  accepted  term. 
Rogers,  Calkins  and  others,  however,  class  them  as  herpetomonads, 
because  of  the  elongated,  flagellated  form  all  take  in  cultures  on  the 
Novy-MacNeal-Nicolle  blood  agar  medium.  It  is,  however,  best  to 
consider  them  as  a  separate  genus,  because  of  their  natural  parasitic 
habits  in  human  beings.  Leveran,  Fantham  and  others  have  shown 
that  it  is  possible  in  the  laboratory  to  induce  the  herpetomonads 
parasitic  in  the  intestine  of  various  insects  to  become  parasitic  in 
various  vertebrates. 

Leishmajiia  donovani  (Kala-azar). — This  parasite  is  the  cause  of 
kala-azar,  a  disease  characterized  by  irregular  fever,  weakness, 
anemia,  cachexia  and  a  remarkable  enlargement  of  the  spleen,  and 
occasionally  of  the  liver.  It  is  chronic,  progressive  and  frequently 
fatal,  the  mortality  being  about  80  to  90  per  cent.  This  disease  is 
common  in  tropical  Asia  and  in  northeastern  Africa. 

Morphology. — The  parasite  is   intracellular,   and   is  found  prin- 


1094 


PATHOGENIC  PROTOZOA 


cipally  in  the  endothelial  cells  of  the  spleen  and  liver,  and  in  the 
bone  marrow.  It  is  oval,  two  to  four  microns  in  diameter,  finely 
granular  and  occasionally  vacuolated.  It  contains  a  large,  round 
nucleus  and  a  smaller  blepharoplast  which  is  oval  or  rod  shaped;  a 
third  body,  a  slender  short  thread,  may  sometimes  be  recognized, 
which  is  presumably  the  undeveloped  flagellum.  Stained  specimens 


4^     ; 


FIG.  144. — LEISHMANIA  DONOVANI.     (Army  Med.  School  Collection, 
Washington,  D.  C.) 

of  blood,  spleen  and  liver  pulp,  and  bone  marrow,  usually  show  large 
endothelial  cells  or  leucocytes  closely  packed  with  parasites,  one  to 
two  hundred  to  a  single  cell.  Multiplication  in  the  body  is  by  simple 
division,  and  incompletely  divided  pairs  of  organisms  are  frequently 
seen.  Cultures  have  been  obtained  in  citrated  blood  and  on  the 
usual  N.  N.  N.  medium.  When  fully  grown  the  cultural  organisms  are 
typical  herpetomonads  (leptomonads)  ;  the  cell  body  elongates  and 
the  rudimentary  whip  develops  into  a  true  flagellum.  Both  dogs  and 
monkeys  are  susceptible  to  artificial  inoculations. 


MASTIGOPHORA  1095 

The  parasite  is  probably  transmitted  by  some  insect,  either  cimex 
(Rogers),  or  by  the  dog  flea,  Ctenocephalus  canis  (Wenyon),  or  a 
plant-feeding  bug,  Conorhinus,  which  occasionally  sucks  blood. 

Animal  Pathogenicity. — Wenyon  in  19138  inoculated  a  dog  with 
splenic  emulsion  from  a  man  who  died  in  London  of  kala-azar  con- 
tracted in  Calcutta.  The  parasite  has  been  successfully  carried 
through  five  animals,  and  in  1915  an  examination  of  the  bone  marrow 
showed  not  only  typical  leishmania,  but  also  a  few  large,  well-marked 
leptomonad  forms.  Similar  forms  were  described  by  Escomel  in  1911, 
from  South  American  dermal  lesions.  Monkeys  may  also  be  infected. 

Leishmania  tropica  (Delhi  or  Aleppo  boil)  is  the  organism  found 
in  a  local  skin  affection  variously  termed  Delhi  boil,  Aleppo  boil  or 
tropical  ulcer.  While  it  is  probably  transmitted  by  some  insect, 
there  is  as  yet  no  definite  proof.  The  incubation  period  is  about 
two  months,  while  the  disease,  once  manifest,  lasts  twelve  to  eighteen 
months  and  is  followed  by  immunity  for  life. 

The  parasite,  which  was  first  described  by  J.  H.  Wright,9  shows 
minor  differences  from  leishmania  donovani,  particularly  a  variable 
morphology,  all  gradations,  from  the  usual  oval  to  elongated  narrow 
forms  with  pointed  ends,  being  found. 

Cultures  may  be  obtained  on  the  N.  N.  N.  blood  agar,  which 
develop  into  leptomonads,  as  with  Leishmania  donovani.  Dogs  and 
monkeys  are  susceptible  to  artificial  inoculation,  and  it  is  possible 
that  in  nature  the  disease  is  carried  from  dogs  to  human  beings  by 
some  insect. 

Leishmania  infantum  (Infantile  Splenomegaly)  was  described  by 
Nicolle  in  1909  from  cases  of  infantile  splenomegaly  occurring  in 
Northern  Africa.  It  is,  however,  not  limited  to  this  region,  but 
occurs  throughout  the  whole  Mediterranean  District.  Javarone10 
described  110  cases  observed  in  Naples  from  1913  to  1920.  The 
disease  resembles  kala-azar  in  all  respects,  except  that  the  patients 
are  young  children,  and  it  is  possible  it  is  the  same  disease.  With- 
out treatment  the  disease,  like  kala-azar  runs  a  progressive  course 
almost  always  leading  to  death.  The  parasites  are  found  in  abun- 
dance in  the  liver,  spleen  and  bone  marrow  at  autopsy  and  may  be 
cultivated  in  the  usual  way  on  the  N.  N.  N.  blood  agar. 


8  Wenyon,  Jour.  Trop.  Med.  and  Hyg.,  London,  1915,  xviii,  218. 

9  Wright,  J.  H.,  Jour.  Med.  Res.  Bost.  1903,  x,  472. 

10  Javarone,  N.,  Infantile  Leishmaniasis  hi  Naples  and  Neighborhood,  Tropical 
Diseases  Bulletin,  London,  1920,  16,  454, 


1096  PATHOGENIC    PROTOZOA 

Animal  Pathogenicity. — The  disease  occurs  naturally  in  African 
dogs,  and  they  are  probably  the  source  of  infection,  the  parasite  being 
carried  by  a  flea  or  some  other  insect.  Dogs,  monkeys  and  guinea-pigs 
are  susceptible  to  artificial  inoculation. 

Treatment. — The  use  of  tartar  emetic  in  the  treatment  of  kala-azar 
is  due  to  the  success  of  the  treatment  of  infantile  leishmaniasis  intro- 
duced by  Di  Christina  and  Caronia.  In  kala-azar  it  has  now  been 


L          r*  -.>*'  J 

FIG.  145. — LEISHMANIA  INFANTUM.     (Army  Med.  School  Collection,  Washington, 

B.C.) 

used  successfully  by  many  and  when  properly  given  is  without  danger 
and  gives  a  large  number  of  recoveries. 

The  untreated  disease  has  a  mortality  of  about  90%.  Dobbs- 
Price11  has  reported  2000  injections  with  67%  of  recoveries.  He  used 
a  1%%  solution  of  sodium  antimony  tartrate,  in  a  weekly  dosage 
increasing  from  1  to  8  c.c. 

11  Dodds-Price,  J.,  Kala-Azar  in  Europeans  in  the  Nowgong  District  of  Assam 
— Indian  Medical  Gazette,  1920,  Vol.  55,  No.  3,  pp.  87-89. 


MASTIGOPHORA  1097 

Since  intravenous  treatment  of  children  with  any  drug  is  difficult, 
a  search  has  been  made  for  preparations  which  can  be  given  into  the 
muscles,  without  producing  pain  or  necrosis.  Some  promising  results 
have  been  obtained  by  Spagnolio  and  Manson-Bahr,12  with  acetyl-p- 
amino-phenyl-stibiate  of  sodium,  and  "stibenyl"  a  related  drug.  The 
drug  is  dissolved  in  distilled  water  and  "stibenyl"  according  to 
Manson-Bahr,  may  be  given  in  doses  up  to  0.6  gram  for  an  adult.  The 
treatments  are  given  weekly  over  a  period  of  4  or  5  months  with 
resulting  cure.  The  time  of  treatment  may  be  shortened  by  using  the 
intravenous  route  when  possible. 

The  prognosis,  as  a  result  of  the  new  treatment  with  antimony, 
may  now  be  considered  good,  particularly  in  acute  cases. 

Two  other  forms  of  dermal  leishmaniasis  have  been  described ;  the 
first,  due  to  Leishmania  braziliensis,  occurs  in  many  parts  of  South 
America.  The  parasite  is  morphologically  identical  with  Leishmania 
tropica.  Since  the  disease  is  always  contracted  in  the  virgin  forest, 
one  name  for  the  affection  is  forest  yaws ;  uta  and  espundia  are  prob- 
ably different  clinical  forms  of  the  same  disease.  The  transmitting 
insect  cannot  well  belong  to  the  household  vermin  or  domestic  insects ; 
sylvan  insects  such  as  the  ixodides,  tabanides,  simulids,  mosquitoes  and 
ConorJiinus  are  all  suspected  of  being  carriers. 

The  second  form  is  called  Leishmania  nilotica  (Brumpt,  1913). 
and  is  found  in  non-ulcerating  keloid  nodules  in  Egyptian  negroes. 
Morphologically,  the  parasite  is  indistinguishable  from  Leishmania 
tropica. 


12  Spagnolio,  Giuseppe,  Tropical  Diseases  Bulletin,  London,  1920,  16,  455,  and 
Manson-Bahr,  Philip,  Brit.  Med.  Jour.  1920,  Aug.  14,  235. 


CHAPTER  LVI 

CLASS  III— SPOROZOA1 

SUB-CLASS— TELOSPORIDIA 

HEMOSPORIDIA 

THE  Hemosporidia  and  Sarcosporidia  are  the  only  members  of  this 
order  of  medical  interest.  The  hemosporidia  belong  to  the  sub-class 
Telosporidia  of  the  Sporozoa,  because  spore  formation  begins  at  the 
end  of  the  life  cycle. 

The  systematists  have  not  yet  agreed  upon  the  proper  classification 
of  this  group  of  parasites;  consequently  the  older  arrangement  will 
be  followed.  They  are,  like  the  coccidia,  parasites  of  cells,  at  least 
during  the  schizogenous  cycle ;  all  change  hosts  to  some  insect  for  the 
sporogenous  cycle.  As  the  name  implies,  they  live  in  blood  cells  and 
are  rapidly  growing  ameboid  bodies,  which,  beginning  as  sporozoites, 
penetrate  the  host  cells  and  develop  into  trophozoites.  These  grow 
rapidly  to  adult  segmenting  parasites,  in  which  case  they  are  called 
schizonts,  or  to  sexual  forms,  or  gametes,  when  they  are  termed 
sporonts.  In  the  course  of  their  development,  most  species  produce 
melanin  from  the  destruction  of  the  hemaglobin. 

The  nucleus,  which  is  readily  stained,  is  single  and  posesses  a 
karyosome ;  the  mature  schizont  divides  into  many  small  forms  called 
merozoites,  and  these,  when  freed  by  the  rupture  of  the  degenerated 
erythrocyte,  escape  into  the  blood  plasma,  and  if  not  phagocyted, 
penetrate  other  erythrocytes  and  repeat  the  asexual  or  schizogenous 
cycle.  The  pigment  and  undivided  portion  (restkorper)  of  the  cyto- 
plasm of  the  mother  cell  accumulate  in  the  bone  marrow,  spleen  and 
other  viscera. 

After  a  number  of  cycles  of  asexual  multiplication  have  been  lived 
through,  a  new  development  takes  place  and  sexual  forms  begin  to 
appear  in  the  circulation.  These  grow  to  large  size,  yet  show  no  indi- 
cation of  division  into  merozoites  and  were  at  one  time  considered 

1  For  classification,  see  page  1049. 

1098 


SPOROZOA  1099 

degeneration  forms.  Two  varieties  may  be  distinguished,  one  with  a 
dark  staining  cytoplasm  and  fine  granular  melanin,  and  the  other  witk 
light  staining,  hyaliii  cytoplasm  and  coarse  pigment;  the  former, 
loaded  with  reserve  food  material,  is  the  female  or  macrogametocyte ; 
the  latter,  the  male  or  microgametocyte.  The  gametes  do  not  develop 
further  until  taken  into  the  digestive  tract  of  the  insect  host.  For 
purposes  of  study,  however,  the  liiicrogametocytes  may  be  made  to 
exflagellate  on  the  slide,  dampened  a  little  by  breathing  upon  it,  to 
stimulate  the  condition  in  the  insect  host.  In  such  a  preparation,  the 
flagella,  or  microgametes,  -may  be  seen  actively  moving  inside  the  cell 
body,  whose  wall  they  ultimately  rupture,  and  all,  four  to  eight,  escape 
and  whip  about  until  they  come  in  contact  with  a  macrogametocyte, 
when  one  microgramete  enters  through  the  micropyle  and  finally  fuses 
with  the  female  nucleus. 

Hemoproteus  columbae  (Halteridium). — This  parasite  of  the  red 
blood  cells  of  doves  was  described  in  1891  by  Celli  and  Sanfelice. 
It  is  widely  distributed  in  nature  and  has  been  reported  from 
Europe,  Asia  and  North  and  South  America.  The  organism  is 
found  within  the  cytoplasm  of  the  erythrocyte;  the  nucleus,  which 
is  not  regularly  displaced,  is  surrounded  by  the  growing  parasite 
like  a  halter,  and  for  this  reason  it  was  named  halteridium  by  Labbe. 
It  is  sluggishly  ameboid  and  produces  an  abundance  of  melanin,  and 
when  the  blood  is  drawn  the  ripe  male  sporonts,  the  microgametocytes, 
rupture  easily,  liberating  the  active  flagella,  or  microgametes.  Under 
favorable  circumstances  the  fertilization  of  the  macrogametocyte  by 
the  microgametes  may  be  observed  on  the  slide,  and  it  was  while  work- 
ing with  this  parasite  that  Macallum  first  followed  out  the  whole 
process  of  fertilization  in  the  hemosporidia  and  gave  the  proper 
explanation  of  the  flagellate  stage  seen  in  the  malarial  parasite. 

In  the  blood  of  the  dove  this  parasite  is  usually  seen  as  a  large  or 
small  crescent,  partly  encircling  the  nucleus;  the  gametes  are  readily 
recognized  by  the  usual  marks,  that  is,  the  female,  or  macrogametocyte, 
is  rich  in  reserve  material  and  the  stained  specimen  takes  a  deep  color ; 
the  male,  or  microgametocyte,  being  poor  in  reserve  material  stored 
in  the  cytoplasm,  appears  relatively  pale  in  stained  specimens. 

The  invertebrate  host  of  the  parasite  is  Lynchia  maura  (Bigot), 
or  Lynchia  lividocolor,  a  biting  hippoboscid  fly  of  louse-like  habits 
which  lives  in  the  nest  and  in  the  plumage.  The  cycle  in  the  fly  has 

2  Adie,  Helen,  Indian  Jour.  Med.  Research,  Calcutta,  1915. 


1100 


PATHOGENIC  POKTOZOA 


been  successfully  worked  out  by  Adie,2  who  has  demonstrated  the 
ookinetes,  zygotes  and  oocysts  in  the  lower  portion  of  the  midgut.    As 


FIG.  146. — H^MOPROTEUS  COLUMB.E.  la  to  3a,  Development  of  female  parasite  in 
blood  of  dove;  Ib  to  36,  Development  of  male  parasite  in  blood  of  dove;  4taf 
46,  56,  6  to  12,  Development  in  the  digestive  tube  of  the  fly  (Lynchia) ;  13  to 
20,  Development  of  the  parasite  inside  leucocytes  in  the  lung  of  the  dove. 
(After  Avagao.  MacNeal,  "Pathogenic  Microorganisms,"  published  by  P. 
Blakiston's  Son  &  Co.) 

the  oocyst  grows,  it  stands  out  from  the  gut  wall  and  finally  shows  the 
striations  indicative  of  the  presence  of  sporozoites;  after  rupture  of 


SPOROZOA  1101 

the  mature  cyst,  these  collect  in  large  numbers  in  the  salivary  glands 
and  ducts. 

The  life  history  of  the  parasite  is  seen  to  be  like  that  of  proteosoma 
and  malaria,  except  that  the  asexual  or  schizogenous  cycle  appears  to 
be  lacking. 

Proteosoma  (plasmodium)  prseoox. — This  parasite  is  a  typical 
representative  of  the  sporozoa,  and  is  interesting  historically,  since 
it  was  the  one  with  which  Ross  worked  in  1898,  when  he  first 
demonstrated  the  part  played  by  the  mosquito  in  "bird  malaria." 

Grassi  and  Feletti  described  the  parasite  in  1890  under  the  name 
of  hemameba  precox.  It  is  widely  distributed  geographically,  and  is 
common  in  the  blood  of  small  birds,  sparrows,  robins  and  larks.  It 
can  be  propagated  in  the  laboratory  in  the  blood  of  canaries  with- 


FIG.  147. — PROTEOSOMA  PKECOX  IN  BLOOD  OF  FIELD  LARK.  A.  Young  parasite  in 
blood  cell;  5,  Half -grown  parasite  which  has  pushed  aside  nucleus  of  blood  cell; 
C,  Parasite  with  clump  of  pigment  and  many  nuclei;  D,  Division  into  many 
merozoites.  (After  Doflein  and  Wasielewski.  MacNeal,  "Pathogenic  Micro- 
organisms," published  by  P.  Blakiston's  Son  &  Co.) 

out  great  difficulty;  sparrows,  however,  do  not  long  survive  in  cap- 
tivity unless  kept  in  round  glass  jars,  where  they  cannot  injure  them- 
selves by  dashing  against  the  walls.  The  blood  for  examination  is 
obtained  from  the  cephalic  wing  vein,  close  to  the  body,  which  is 
nicked  with  a  razor,  and  the  blood  taken  up  in  a  capillary  glass  tube 
containing  a  little  citrate  solution.  To  inoculate  a  new  bird,  it  is 
sufficient  to  inject  a  small  quantity  of  citrated  blood  from  an  infected 
canary  into  the  breast  muscles  of  the  new  bird,  transferring  to  a  new 
host  at  intervals  of  a  month  or  less.  Because  it  is  not  difficult  to 
keep  on  hand,  this  organism  may  be  used  for  class  study  in  localities 
where  malarial  cases  are  infrequent.  There  is  no  apparent  reason 
for  placing  it  in  a  different  genus  from  the  malarial  parasites. 

The  entire  asexual  cycle,  schizogony,  may  be  studied  in  the  periph- 
eral circulation,  as  in  quartan  malarial  fever. 

In  nature  it  is  transmitted  by  both  culex  and  stegomyia  (Aedes 


1102  PATHOGENIC  PROTOZOA 

calopus),  and  its  development  is  briefly  as  follows:  The  bird  is 
inoculated  by  the  mosquito  with  spindle-shaped  young  forms  known  as 
sporozoites.  These  possess  the  power  of  ameboid  motion,  and  rapidly 
penetrate  into  an  erythrocyte,  in  which  they  grow  quickly ;  they  con- 
stantly move  about  inside  the  cell  until  nearly  full  grown,  and  are 
during  this  stage  called  trophozoites.  The  substance  of  the  ery- 
throcyte is  rapidly  consumed  by  the  parasite  and  a  dark  pigment, 
melanin  or  hemozoin,  is  formed  from  the  destroyed  hemaglobin.  The 
mature  parasite  divides  into  many  small  forms  called  merozoites.  and 
these,  when  freed  by  the  rupture  of  the  degenerated  erythrocyte, 
escape  into  the  blood  plasma,  and  if  not  phagocyted,  penetrate  other 
erythrocytes  and  repeat  the  asexual  or  schizogenous  cycle.  The  pig- 


FlG.   148. MlDGUT  OF  CULEX  MOSQUITO,  COVERED  WITH  OoCYSTS  OF    PROTEOSOMA 

PRECOX.     V,  VASA  MALPIGHII.     (After  Doflein  and  Ross.     MacNeal,  "Path- 
ogenic Microorganisms,"  published  by  P.  Blakiston's  Son  &  Co.) 

ment  and  undivided  portion   (restkorper)   of  the  cytoplasm  of  the 
mother  cell  accumulate  in  the  bone  marrow,  spleen  and  other  viscera. 


MALARIA 

This  is  one  of  the  most  common  and  widespread  of  preventable 
human  diseases,  and  in  some  localities  is  the  cause  of  a  greater  mor- 
tality and  morbidity  than  tuberculosis.  It  is  caused  by  one  or  more 
of  the  three  forms  of  the  malarial  plasmodium.  As  a  rule  the  infec- 
tions are  simple,  yet  in  the  tropics  it  is  not  uncommon  to  find  two 
or  even  three  species  of  plasmodia  in  the  same  patient,  and  this 
condition  is  called  a  mixed  infection. 

History. — The  disease  under  various  names,  as  chills  and  fever, 
Roman  fever,  Chagres  fever,  has  been  known  since  the  greatest 
antiquity.  The  cause  was  not  discovered  until  1880,  when  Laveran,  a 
French  military  surgeon  stationed  in  Algeria  first  saw  the  organism 
and  described  it  as  the  cause  of  malaria.  He  saw  and  described  ;oof 


SPOROZOA  1103 

only  the  pigmented  trophozoite,  but  also  the  crescentic  gametocytes  and 
flagellating  microgametes,  and,  because  of  the  activity  of  the  flagella, 
called  the  parasite  Oscillaria  malaria,  a  name  afterwards  given  up. 
Later,  in  1885,  Celli  and  Marchifava  described  the  parasite  with 
greater  accuracy  and  named  it  Plasmodium  malarias,  a  poor  name, 
since  it  describes  merely  a  condition  assumed  by  some  fungi  and 
mycetozoa,  yet,  according  to  the  rules  of  zoological  nomenclature,  it 
must  stand.  In  the  same  year,  Golgi  described  the  quartan  parasite 
and  in  the  following  year  demonstrated  the  relation  of  the  various 
stages  of  the  life,cycle  of  the  tertian  parasite  to  the  temperature  .curve. 

Even  in  antiquity  many  had  noted  the  curious  distribution  of 
malaria,  and  its  intimate  relation  to  swamps  and  marshy  places. 
Manson,  who  had  already  shown  the  role  played  by  an  infected  mos- 
quito in  transmitting  filarial  disease,  in  1894,  suggested  that  the 
epidemiology  of  the  disease  could  best  be  explained  by  the  hypothesis 
that  it  was  conveyed  by  the  bite  of  some  blood-sucking  insect,  probably 
the  mosquito. 

For  years  the  interpretation  of  the  flagella  was  a  subject  of  con- 
troversy. They  were  regarded  as  degeneration  products  by  some  and 
as  living  elements  by  others.  In  1897,  MacCallum,  working  with 
halteridium,  was  able  to  show  that  they  were,  in  fact,  spermatozoa,  as 
he  saw  them  penetrate  and  fertilize  the  macrogametes,  or  large 
spherical  forms  without  flagella. 

In  1897,  Ross,  of  the  British-  Indian  Medical  Service,  described  the 
beginning  of  the  sporogenous  cycle  in  what  he  called  a  dapple-winged 
mosquito,  which  we  now  recognize  as  an  anopheline.  Following  out 
further  Manson 's  hypothesis,  he  was  able  the  same  year  after  long  and 
laborious  research  to  clear  up  the  method  of  transmission  of  bird 
malaria,  proteosoma,  an  analogous  disease.  Grassi  and  Bignami  and 
Bastianelli,  in  1898,  succeeded  in  demonstrating  the  complete  life 
cycle  of  the  human  form  of  malaria  in  the  anopheles  mosquito. 

Geographical  Distribution. — The  disease  is  found  in  a  belt  round 
the  world  extending  from  40  degrees  S.  latitude  to  60  degrees  N. ;  it  is, 
however,  not  equally  distributed  throughout  this  zone,  and  even  in  the 
tropics  there  are  many  malaria-free  areas,  principally  in  the  regions 
of  higher  altitudes,  since  the  special  home  of  malaria  is  in  the  low- 
lying,  swampy  and  torrid  coastal  districts  and  river  basins.  Islands 
at  a  distance  from  the  main  land  may  be  entirely  free.  Malaria 
reaches  its  maximum  intensity  in  the  tropics,  where  the  anopheline 
mosquitoes  breed  continuously  throughout  the  year-  and  new  infec- 


1104 


PATHOGENIC  PROTOZOA 


tions  may  occur  at  any  time  ;  while  in  the  sub-tropics  and  temperate 
regions  it  is  a  seasonal  disease,  appearing  soon  after  the  onset  of  hot 
weather  with  its  new  crop  of  anophelines  and  continuing  until  the 
first  cold  weather  which  destroys  most  of  the  infected  mosquitoes. 
It  is  possible  that  the  disease  may  be  carried  over  from  season  to 
season  by  the  hibernating  mosquito  although  definite  proof  of  the 
importance  of  this  is  lacking.  It  is  carried  over  to  the  next  season 
by  the  human  carrier,  in  whom  the  disease  may  be  latent  or  who  may 
have  suffered  from  clinical  relapses  throughout  the  year.  Modern 
times  have  seen  it  disappear  from  many  regions  where  it  was  formerly 


N$^!c^0 


FIG.    149.  —  PLASMODIUM   VIVAX 
(Army    Med.  School  Collection, 
Washington,  D.  C.) 


FIG.  150.  —  PLASMODIUM  VIVAX.  (Gamete.) 
(Army  Med.  School  Collection,  Washing- 
ton,  D.  C.) 


endemic,  because  of  increased  cultivation  of  the  soil  and  better  surface 
drainage,  as,  for  example,  in  England  and  the  Ohio  river  valley. 

In  the  registration  area  of  the  United  States  there  were  1565 
deaths  from  malaria  in  1913  ;  in  Italy,  up  to  1900,  the  average  number 
of  deaths  from  this  cause  annually  was  16,000.  One  cannot  obtain  a 
true  picture  of  the  importance  of  the  disease,  however,  from  mortality 
statistics,  since  it  is  not  often  fatal,  and  the  morbidity  is  out  of  pro- 
portion to  the  mortality.  In  many  villages,  where  it  is  endemic,  one- 
third  to  one-half  the  population  may  have  parasites  in  the  blood,  most 
of  them  without  clinical  symptoms,  yet  they  are  not  able  to  work  and 
the  children  remain  undeveloped  and  backward.  Much  of  the  illness 
attributed  to  hookworm  infection  is,  in  reality,  due  to  latent  malaria. 


SPOIIOZOA  1105 

During  the  first  part  of  the  acute  attack  the  predominant  forms  are 
sehizonts,  but  later  in  the  course  of  the  disease,  as  a  result  of 
increasing  immunity  or  of  treatment,  the  sporogenous  cycle  becomes 
evident.  The  trophozoites,  in  increasing  numbers,  develop  into 
sporonts  rather  than  schizonts.  These  forms  do  not  develop  a  vacuole 
but  increase  in  size  and  remain  round  or  oval  bodies.  When  mature 
they  occupy  most  of  the  red  cell  but  show  no  sign  of  nuclear  change 
or  of  segmentation  and  they  are  then  called  gametocytes.  The  sex 
can  be  distinguished  in  well  stained  specimens  by  remembering  that 
the  micro gametocyte,  or  male  form,  is  rich  in  nuclear  chromatin, 
of  which  the  flagella  or  spermatozoa  will  be  formed  after  the  game- 
tocyte has  been  ingested  by  the  proper  mosquito.  The  macro  game- 
tocyte, or  female  form,  is  distinguished,  on  the  other  hand,  by  the 
store  of  nutrient  material  in  the  cytoplasm,  causing  it  to  stain 
deeply. 

After  ingestion  by  a  susceptible  mosquito  the  red  cells  undergo 
dissolution  and  the  contained  gametocytes  are  liberated.  The  micro- 
gametocyte  sends  out  several  micro  gametes  (flagella)  one  of  which 
fertilizes  a  macrogamete  by  penetrating  the  cytoplasm  and  uniting 
with  its  nuclear  chromatin.  The  fertilized  cells  change  its  shape 
from  round  to  ovoid  and  becomes  motile  and  is  called  a  traveling 
vermicule,  or  ookinette.  It  travels  to  the  stomach  wall  which  it 
penetrates  and  comes  to  rest  on  its  outer  surface  and  is  then  called 
an  ob'cyst  or  zygate.  The  cyst  or  zygate  increases  in  size  with 
each  successive  segmentation  of  its  nucleus  until  it  is  ripe.  It  then 
ruptures  and  discharges  a  mass  of  sporozoites  into  the  body  cavity. 
These  wander  to  all  parts  of  the  body  of  the  mosquito  but  many 
reach  the  salivary  glands  and  later  the  saliva,  and  when  next  the 
mosquito  bites  they  are  injected  into  the  new  host. 

In  the  state  of  Mississippi  Bass  has  shown  that  the  age  distribution 
is  such  that  23  per  cent  of  the  population  under  20  years  of  age 
showed  parasites  in  the  blood,  whereas  only  19  per  cent  of  persons 
over  20  years  were  infected,  and  that  the  five-year  period  from  5  to 
9  years  of  age  showed  the  greatest  number  of  infections. 

Eace  and  color  are  important,  and  negroes  showed  36  per  cent 
more  infections  than  whites,  and  the  high  point  is  reached  much 
earlier  in  black  than  in  white  children.  (South  Med.  Jour.,  1919, 
12,  456.) 

The  parasites  belong  to  the  class  of  hemosporidia,  and  are  closely 
related  to  the  coccidia,  which  are  parasites  of  epithelial  cells,  while 


1106  PATHOGENIC   PROTOZOA 

the  plasmodia  are  parasitic  on  red-blood  cells.  There  are  two 
divisions  of  the  life  cycle;  that  which  occurs  in  man,  the  endogenous, 
asexual  or  schizogenous,  and  that  which  occurs  in  the  mosquito, 
the  exogenous,  sexual  or  sporogenous ;  for  this  reason  the  mosquito 
is  the  definitive  and  the  man  the  intermediate  host. 

Although  the  life  cycle  of  the  parasite  varies  in  details  in  the 
different  forms  of  malaria,  certain  stages  are  common  to  all,  and  in 
general  the  life  cycle  is  usually  described  as  follows:  an  infected 
mosquito  bites  a  warm  blooded  animal,  often  a  human  being,  in 
order  to  obtain  its  meal  of  blood.  As  it  bites,  it  infects  the  wound 
with  its  saliva  which  contains  sporozoites  coming  from  the  salivary 
glands.  The  sporozoites,  in  the  new  host,  soon  gain  access  to  the 
blood  and  attach  themselves  to  the  erythrocytes  upon  which  they 
become  parasitic.  In  shape  they  are  long  and  slender  spindles,  and 
when  stained  with  the  usual  eosin-methylene  blue  dyes,  show  a  blue 
cytoplasm  and  a  compact  dot  of  red  nuclear  chromatin  in  the  center. 
When  the  sporozoite  escapes  the  phagocytes,  and  succeeds  in  es- 
tablishing itself  on  a  red  cell  it  soon  changes  its  shape  to  the  ring 
form  and  grows  rapidly  and  during  this  growing  stage  is  known 
as  a  trophozoite.  This  last  named  form  may  develop  in  either  of 
two  ways:  in  the  asexual  or  schizogenous  cycle  when  it  is  called 
a  schizont,  or  in  the  sexual  or  sporogenous  cycle,  when  it  is  called 
a  sporont. 

The  schizont  goes  on  to  full  development  in  the  human  host ;  the 
sporont  cannot  complete  its  cycle  until  taken  into  the  stomach  of 
a  suitable  anopheline  mosquito,  capable  of  conveying  malaria. 

The  trophozoit  which  ends  its  life  as  a  schizont  grows  rapidly 
at  the  expense  of  the  red  cell  and  develops  a  characteristic  vacuole 
which  increases  the  area  of  the  parasite  in  contact  with  the  host 
cell.  When  mature  its  nucleus  undergoes  mitotic  changes  and  the 
parasite  divides  into  a  more  or  less  definite  number  of  segments 
called  merozoites ;  these  when  liberated  by  the  disintegration  of  the 
host  cell,  attack  new  erythrocytes  and  develop  during  the  second 
and  subsequent  generations  in  the  same  manner  as  the  sporozoite. 

There  are  three  well-recognized  forms  of  the  plasmodia,  (1)  Plas- 
modium  vivax  (Grassi  and  Filetti),  causing  tertian  fever  (also  called 
"benign  tertian");  (2)  Plasmodium  malarice  (Laveran),  causing 
quartan  fever ;  (3)  Plasmodium  falciparum  (immaculatum)  (Welch), 
causing  the  tropical  form  of  malaria,  the  so-called  aestivo-autumnal 
or  subtertian.  As  the  details  of  development  cannot  be  made  out 


SPOROZOA 


1107 


easily  in  fresh  specimens,  the  following  description  applies  to  those 
stained  with  some  form  of  the  Romanowski  stain. 

Plasmodium  vivax. — The  parasite  of  tertian  fever  has  a  life  cycle 
lasting  forty-eight  hours  arid  is  easily  recognized  only  when  full 
grown,  that  is,  twenty-four  to  forty-eight  hours  after  the  chill. 
While  a  diagnosis  may  be  made  on  younger  forms,  it  is  not  so  readily 
made.  As  its  name  implies,  the  Plasmodium  vivax  is  actively  ame- 
boid, and  pseudopods  and  irregular  outlines  characterize  the  well- 
grown  parasite;  the  infected  erythrocyte  is  swollen,  often  to  twice 


FIG.  151. — PLASMODIUM  VIVAX,  AN  ATYPICAL 
MACROGAMETOCYTE.  Form  interpreted 
by  Schaudinn  as  undergoing  partheno- 
genesis. (Army  Med.  School  Collection, 
Washington,  D.  C.) 


FIG.  152. — PLASMOD'IUM  VIVAX 
(Army.  Med.  School  Collection, 
Washington,  D.  C.)  • 


its  normal  size,  the  hemoglobin  is  pale  and,  especially  in  spreads 
in  which  Hanson's  stain  has  been  used,  it  is  so  much  paler  than 
in  the  surrounding  cells  that  the  infected  cell  stands  out  clearly. 
The  part  of  the  cell  unoccupied  by  the  parasite  is  stippled,  that  is, 
dotted  with  reddish  granules  called  Schuffner's  dots,  and,  as  the 
swollen  red  cell  and  Schuffner's  dots  are  found  in  no  other  form 
of  malaria,  their  presence  is  pathognomoriic  of  tertian. 

The  youngest  form,  the  free  merozoite,  is  rarely  seen,  but  young 
comet-like  forms  composed  of  a  particle  of  red  chromatin  and  a 
little  blue  cytoplasm  may  readily  be  detected  at  the  height  of  the 
fever;  that  is,  a  few  hours  after  the  chill  and  sporulation.  The 
round,  young  schizont  as  it  grows  develops  early  a  central  vacuole 


1108 


PATHOGENIC   PROTOZOA 


and  assumes  the  shape  of  a  signet  ring,  the  red  chromatin  dot  being 
the  stone.  This  small  tertian  ring  grows  rapidly  as  the  fever  sub- 
sides, and  at  the  same  time  the  infected  cell  increases  in  size. 
Twenty-four  hours  after  the  chill  the  ring  has  grown  so  much  that 
it  is  referred  to  as  the  large  tertian  ring,  and  its  tendency  to 
irregularities  of  shape  and  ameboid  form  becomes  apparent,  and 
fine  granules  of  pigment,  called  melanin  or  hemozoin,  begin  to  be 
visible.  After  thirty-six  hours  the  rings  will  all  have  grown  into 
large  ameboid  forms.  After  about  forty  hours  the  parasite  occupies 
almost  the  entire  cell  and  the  pigment  begins  to  collect  in  masses 


FIG.  153.— PLASMODIUM  VIVAX.     (Army  Med.  School  Collection,  Washington,  D.  C.) 

toward  the  center.  Soon  after  the  first  signs  of  segmentation  appear, 
which  becomes  more  and  more  distinct  until  fifteen  to  twenty 
separate  segments  or  merozoites  are  seen,  each  composed  of  nucleus  and 
cytoplasm.  The  pigment  of  the  adult  parasite  and  the  unused 
portion  of  the  cytoplasm  are  cast  off  after  segmentation  as  a  rest- 
korper,  which  is  promptly  phagocyted  and  such  masses  accumulate 
in  the  spleen,  bone  marrow  and  viscera.  With  rupture  of  the 
erythrocyte,  at  the  time  of  the  chill,  the  merozoites  are  set  free, 
and  if  not  phagocyted,  immediately  attack  new  erythrocytes  and 
the  asexual  or  schizogenous  cycle  is  repeated,  until  treatment  or 
Jncreasing  immunity  halts  or  alters  the  cycle. 


SPOROZOA  1109 

In  practice  it  is  not  unusual  to  find  parasites  of  different  ages 
in  the  same  film,  as  some  individuals  seem  to  develop  in  advance 
of  others;  in  this  case,  however,  there  will  not  be  much  difference 
in  their  appearance.  When  extreme  difference  of  age  is  noted  in 
films  it  is  probable  that  there  have  been  several  different  inoculations, 
producing  double  or  triple  infections  with  quotidian  or  irregular 
fever  curves,  and  such  cases  are  not  uncommon. 

As  all  the  forms  so  far  described  belong  to  the  schizogenous 
cycle,  they  may  be  called  schizonts,  or  trophozoites  of  the  schizo- 


I 


FIG.  154. — PLASMODIUM  VIVAX.     (Army  Med.  School  Collection,  Washington,  D.C.) 

geiious  cycle.  The  sporogenous  cycle  begins  in  man  and  is  com- 
pleted in  the  mosquito.  The  earliest  sexual  forms  noted  were  the 
so-called  "spheres,"  large  adult  parasites,  first  seen  in  wet  prepara- 
tions, which  did  not  segment  with  the  schizonts.  They  are  now 
called  gamctocytes  and  after  the  disease  has  lasted  some  time  are 
found  in  films  made  at  all  stages  of  the  fever;  that  is,  they  are 
incapable  of  further  development  until  taken  into  the  stomach  of 
the  mosquito.  The  possibility  of  parthenogenesis  will  be  referred 
to  later.  In  appearance  they  are  round  or  oval,  and  m  this  fever 
may  be  twice  the  si/e  of  the  red  cell.  As  a  rule  a  narrow  margin 
of  red  cell  is  visible  after  Romaiiowski  stains,  although  the  gamete 


1110  PATHOG  U  N 1 C   PK( >T< >Z( ) A 

may  lie  free  in  the  plasma.  Unlike  the  schizonts,  the  gamctocytcs 
have  the  pigment  uniformly  distributed  throughout  the  body  and 
there  is  no  indication  of  segmentation.  The  young  sporonts  are 
distinguished  from  schizonts  by  the  absence  of  the  vacuole,  and, 
when  a  little  older,  by  a  larger  amount  of  hemozoin. 

Flasmodium  malarias. — The  quartan  parasite  has  a  life  cycle  of 
seventy-two  hours,  or  twenty-four  hours  longer  than  the  tertian,  and 
the  paroxysms  come  on  every  third  day,  or,  according  to  the  Italian 
method  of  reckoning  time,  on  the  fourth  day.  The  young  rings  of 
the  plasmodium  mala-rice  are  indistinguishable  from  young  tertian 
rings,  but  the  diagnosis  may  be  made  on  older  forms.  The  bleach- 


FIG.    155.  —  PLASMODIUM    M  A  L  A"R  i  m  .      FIG.  156— PLASMODIUM  MALARLE.  (Army 
(Army  Med.  School  Collection,  Wash-  Med.  School  Collection,  Washington, 

ington,  D.  C.)  D.  C.) 

ing,  enlargement  and  stippling  of  the  erythrocyte  character- 
istic of  tertian  is  never  found  in  quartan  fever,  the  infected  ery- 
throcyte being  almost  normal  in  appearance.  The  well-grown 
quartan  parasite  does  not  show  amoebic  changes  but  assumes  a  band 
form,  more  or  less  wide,  stretching  across  the  red  cell  from  border 
to  border;  with  increasing  age  the  band  widens  until  the  parasite 
is  nearly  square  and  the  hemozoin  accumulates  toward  the  center. 
Segmentation  gives  rise  to  almost  symmetrical  "daisy"  forms,  show- 
ing six  to  eight  or,  rarely,  fourteen  merozoites.  Parasites  of  dif- 
ferent ages  may  be  found,  as  in  tertian,  and  it  is  characteristic  of 
quartan  fever  that  examples  of  all  stages  of  the  life  cycle  may  be 
found  at  the  proper  time  in  the  peripheral  circulation.  Gametocytes 
differ  from  tertian  mainly  in  size,  since  they  are  never  larger  than 


SPOROZOA 


1111 


the  normal  erythrocyte  until  after  the  latter  has  raptured,  but  when 
free  in  the  plasma  it  is  practically  impossible'  1o  distinguish  them 
from  tertians. 

Plasmodium  falciparum. — The  parasite  of  a^stivo-autumnal  fever, 
I'lfisnifHlinm  falciparum,  differs  considerably  from  the  two  forms  al- 
ready described;  the  life  cycle  varies  between  twenty-four  and  forty- 
eight  hours,  and,  at  least  in  new  infections,  only  ring  forms  are 
found  in  the  peripheral  blood,  although  at  a  later  stage  crescentic 
gametocytes  may  be  present.  The  youngest  aestlvo-autumnal  rings, 
found  at  the  height  of  the  fever,  are  more  delicate  than  the  younp; 
tertians.  As  the  temperature  falls  the  rings  increase  in  size,  but 


FIG.  157. — PLASMODIUM  MALARLE. 
(Army  Med.  School  Collection,  Wash- 
ington, D.  C.) 


FIG.  158.  —  PLASMODIUM  FALCIPARUM. 
(X  150.0)  (Army  Med.  School  Col- 
lection, Washington,  D.  C.) 


without  change  of  form;  the  growth  is  not  uniform,  but  occurs  as 
a  thick  crescentic  swelling  on  the  convex  surface  of  the  ring,  and 
occasionally  more  than  one  such  swelling  is  present.  The  large 
aestivo-autumnal  ring,  found  after  the  febrile  paroxysm  has  passed, 
occupies  one-third  to  one-half  the  red  cell,  which  is  never  swollen 
nor  stippled,  as  in  tertian,  and  the  parasite  is  never  band-like,  as 
in  quartan.  Segmenting  parasites  are  almost  never  seen  in  the 
peripheral  blood  in  aestivo-autumnal  fever,  though  in  tertian  they  are 
common  and  in  quartan  numerous.  If,  however,  films  are  prepared 
at  autopsy  from  the  spleen,  liver,  bone  marrow  and  brain,  enormous 
numbers  of  segmenting  forms,  together  with  all  other  stages  of  the 
parasite,  may  be  found.  The  full  grown  segment  cr  occupies  one- 
third  to  one-half  the  cell  and  shows  a  collection  of  hemozoin  in  large 


1112 


PATHOGENIC   PROTOZOA 


blocks  in  the  center.  The  merozoites  vary  in  number  from  eight 
to  twenty-five.  In  addition  to  the  small  and  large  rings  the  per- 
ipheral blood  shows,  after  the  fever  has  lasted  sufficiently  long,  the 
sexual  forms  or  gametocytes.  The  infected  erythrocyte  is  never 
stippled  nor  swollen,  but,  on  the  contrary,  may  appear  shrunken. 
Both  the  micro-  and  macrogametocytes  in  aestivo-autumnal  fever  are 
crescentic  in  shape,  their  length  being  about  one  and  one-half  and 
the  width  about  one-half  that  of  an  erythrocyte;  the  pigment  is 
collected  toward  the  center,  which  is  rather  paler  in  stained  speci- 
mens than  the  poles.  At  first  sight  the  gametocytes  appear  to  lie 
free  in  the  plasma,  yet  in  stained  specimens  a  rim  or  rib  of  the  pale 

red  cell  may  be  seen  on  the  con- 
cave side.  When  liberated  from 
the  erythrocyte  the  gametocyte 
becomes  first  spindle-shaped  and 
finally  oval  or  round.  The  male 
crescent  is  short  and  broad,  and 
the  female  relatively  long  and 
slender. 

The  Finer  Structure  of  the  Plas- 
modia. — The  finer  details,  which 
are  only  hinted  at  in  fresh  speci- 
mens and  in  those  stained  with  Man- 
son  's  stain,  can  be  studied  to  ad- 
vantage in  those  stained  with  some 
one  of  the  many  modifications  of 
the  Romanowski  stain,  such  as  that 
of  Wright,  Hastings,  MacNeal  or  Giemsa. 

The  tertian  parasite,  which  lies  in  a  red  cell,  may  be  seen  to  be 
divided  into  a  blue  cytoplasm  and  a  brilliant  red  nucleus,  and  it 
would  be  well  for  the  novice  to  remember  that  these  three  conditions 
must  be  satisfied  before  the  diagnosis  of  malaria  can  be  made ;  the 
principal  stumbling-block  is  the  blood  platelet,  often  found  overlying 
a  red  cell,  but  it,  although  possessing  a  ragged  blue  cytoplasm,  has 
always  a  relatively  large  purple  nucleus.  The  chromatin  of  the 
young  rings  is  usually  present  as  a  single  dot,  but  two  such  dots 
are  frequently  seen.  In  older  forms  of  the  tertian  and  quartan 
parasites  the  various  changes  found  in  mitosis  may  be  followed  in 
nucleus.  The  whole  schizogenous  cycle  may  be  followed  by 


FIG.  159. — PLASMODIUM  FALCIPARUM. 
(X1500.)  (Army  Med.  School  Col- 
lection, Washington,  D.  C.) 


the 


taking  blood  smears  from  a  single  case  of  malaria  at  intervals  of 


SPOROZOA 


1113 


three  or  four  hours  for  forty-eight  hours  for  tertian  and  sestivo- 
autumnal,  and  for  seventy-two  hours  for  quartan. 

Two  forms  of  sporonts  or  gametocytes  may  be  seen;  in  one  the 
quantity  of  chromatin  is  large  and  the  cytoplasm  pale  blue ;  while 
in  the  other  the  reverse  is  found,  the  nuclear  chromatin  is  com- 
paratively small  in  quantity  and  the  cytoplasm,  being  rich  in 
nutrient  material,  stains  deeply.  The  first  form,  with  abundant 
chromatin,  is  the  male,  or  microgametocyte,  and  the  latter  the 


FIG.  160. — PLASMODIUM  FALCIPARUM,  MALE  CRESCENT.     (Army  Med.  School  Col- 
lection, Washington,  D.  C.) 

female,  or  macrogametocyte.  The  differentiation  between  schizont 
and  sporont  may  be  made  while  the  parasites  are  still  quite  young, 
since  the  schizont  is  characterized  by  the  presence  of  a  nutrient 
vacuole,  and  the  sporont,  of  equal  age,  while  lacking  the  vacuole, 
shows  a  greater  amount  of  hemozoin,  which  is  never  concentrated 
in  the  center  of  the  parasite  but  is  scattered  equally  throughout  the 
body.  The  cytoplasm  of  the  sporont  is  less  fluid  than  that  of  the 
schizont  and  shows  no  tendency  to  ameboid  motion.  The  chromatin 
is  relatively  large  in  amount  and,  although  broken  up  more  or  less 


1114  PATHOGENIC   PROTOZOA 

into  granules  and  threads,  shows  no  real  tendency  to  segment  or 
disperse,  but  remains  a  compact  mass. 

The  quartan  parasite,  when  stained  with  Wright's  or  a  similar 
preparation,  shows  quite  regular  and  symmetrical  segmentation, 
usually  into  eight  merozoites.  The  distinction  between  schizont  and 
sporont  and  between  male  and  female  gametocytes  may  be  made 
on  the  same  grounds  as  in  tertian. 

In  aestivo-autumnal  fever  the  chromatin  dot  in  the  young  ring 
is  often  doubled,  or  even  trebled,  and  in  general  is  large  and  stains 
brilliantly.  The  adult  and  half-grown  gametes  may  be  differentiated 
into  male  and  female  by  the  criteria  already  given. 

The  Examination  of  Fresh  Blood. — Directions  have  already  been 
given  (Chap.  LIX)  for  making  wet  preparations  and  if,  by  ringing 
the  cover-glass  with  vaseline,  drying  be  prevented,  the  preparations 
will  keep  and  may  be  studied  for  hours.  In  tertian  fever  the  young 
ring  forms  are  at  first  difficult  to  detect,  unless  the  amount  of  light 
going  through  the  microscope  be  cut  down  to  the  minimum.  As  the 
parasite  grows  older,  an  increasing  number  of  hemozoin  granules 
appear,  and  since  they  are  in  constant  motion  the  parasite  is  readily 
detected.  Its  cytoplasm  is  delicate,  and  with  very  young  parasites 
is  difficult  to  distinguish  from  the  red  cell  itself;  older  parasites, 
however,  develop  pseudopods,  which  are  constantly  projected  and 
retracted,  and  the  entire  organism  shows  active  movements,  render- 
ing it  easy  to  see.  The  pigment  continues  to  increase,  and  in  the 
gametes  is  abundant  and  in  constant  motion ;  the  gametes,  however, 
fail  to  show  any  ameboid  changes,  and  the  protoplasm  is  stiff  and 
rigid  with  a  regular,  unbroken  margin.  At  times  a  clear  refractile 
spot  is  seen,  which  is  the  nucleus.  The  infected  erythrocyte  is  pale 
and  swollen.  Even  in  unstained  preparations  the  sexes  may  be 
distinguished;  the  microgametocyte  is  about  the  size  of  a  red  cell, 
the  cytoplasm  is  hyalin,  and  after  the  preparation  has  been  made 
ten  to  twenty  minutes  the  flagella,  or  microgametes,  may  be  seen 
thrashing  about  in  the  parasite.  After  repeated  attempts  four  to 
eight  microgametes  rupture  the  cell  and  emerge.  The  macrogamete 
is  larger  than  a  red  cell  and  is  finely  granular  and  no  exflagellatioii 
of  microgametes  occurs. 

In  quartan  malaria  the  differences  already  described  in  stained 
blood  may  be  easily  followed. 

In  aestivo-autumnal  fever  the  diagnosis  with  fresh  blood  is  much 
more  difficult  in  new  infections  because  of  the  relative  scarcity  of 


SPOROZOA  1115 

the  parasites  in  the  peripheral  blood  and  the  exceedingly  small  size 
of  the  young  rings,  the  absence  of  hemozoin  in  them,  and  the  very 
slight  ameboid  motion.  The  older  rings  arc  larger,  contain  some 
pigment  and  are  more  easily  seen.  The  infected  crythrocyte  is 
never  pale  nor  swollen,  but,  011  the  contrary,  may  be  shrunken  and 
brassy  in  color.  The  crcscentic  gametocytes  are  readily  detected, 
and  the  sexes  may  be  differentiated  by  their  shape  and  the  hyaline 
or  granular  character  of  the  cytoplasm. 

Incubation  Period  of  the  Malarial  Fevers. — Two  methods  have 
been  used  to  determine  this  point — the  injection  of  infected  malarial 
blood,  and  biting  experiments  with  infected  anophelines.  By  the 
first  method  the  incubation  period  was  eighteen  days  (the  longest) 
for  quartan,  three  days  (the  shortest)  for  aestivo-autumnal,  and  ten 
days  for  tertian.  By  the  second  method  aestivo-autumnal  was  nine 
to  twelve  days,  tertian  fourteen  to  .nineteen  days,  and  quartan 
eighteen  to  twenty-one  days.  Since  aestivo-autumnal  is  the  only 
parasite  which  can  complete  its  cycle  in  twenty-four  hours,  the  short 
incubation  period  is  easily  understood;  on  the  other  hand,  the  long 
life  cycle  of  quartan,  seventy-two  hours,  explains  its  slower  develop- 
ment. 

Clinical  descriptions  of  the  malarial  fevers  may  be  found  in  the 
standard  text-books  on  medicine,  and  it  is  only  necessary  here  to 
refer  briefly  to  the  various  forms  found  in  practice.  The  classical 
malarial  fever  consists  of  a  series  of  paroxysms,  following  one  an- 
other with  a  definite  periodicity,  daily,  every  other  day,  or  every 
third  day.  Each  paroxysm  is  ushered  in  by  a  pronounced  chill, 
which  is  sometimes  preceded  by  malaise,  headache  and  lassitude. 
The  chill  lasts  from  ten  minutes  to  an  hour  or  more,  and  the  patient 
wraps  himself  up  in  heavy  blankets.  During  the  chill  the  tempera- 
ture begins  to  rise  and  within  a  few  hours  reaches  its  high  point, 
103°  to  106°,  and  then  falls  slowly  to  normal  during  the  next  few 
hours.  The  decline  of  the  fever  is  accompanied  by  a  profuse  per- 
spiration. Successive  paroxysms  may  occur  at  exactly  the  same 
hour  of  the  day,  or  may  anticipate,  febris  anteponens,  or  be  delayed 
an  hour  or  more,  febris  postponens.  The  sequence  of  events,  therefore, 
in  a  typical  malarial  paroxysm  is  malaise,  chill,  fever  and  sweat, 
followed  by  a  period  of  apparent  well-being. 

There,  is,  of  course,  a  typical  symptomatology  and  clinical  course 
in  the  various  forms  of  malarial  fever,  but  it  must  not  be  forgotten 
that  there  are  many  atypical  cases,  and  that  malaria  is  a  protein 


1116  PATHOGENIC   PROTOZOA 

disease,  mimicking  many  other  infections,  and  that  without  a  proper 
examination  of  the  blood  that  a  correct  diagnosis  is  frequently  im- 
possible. Pernicious  malaria  is  often  mistaken  for  typhoid,  for 
gastric,  renal  or  cerebral  disease,  and  malaria  in  children  may  end 
fatally  without  presenting  any  of  the  classical  symptoms.  Blood 
examinations  in  all  febrile  cases  in  malarious  districts  are  therefore 
necessary  to  a  correct  diagnosis. 

The  most  important  sequel  is  marked  secondary  anaemia,  which 
comes  on  rapidly,  even  in  cases  so  mild  that  their  nature  is  unsus- 
pected; in  severe  cases  the  red  cell  count  may  drop  to  three,  two 
or  even  one  million  red  cells  per  cubic  millimeter.  The  occurrence 
of  anemia,  in  malarious  regions,  should  always  lead  one  to  suspect 
this  disease.  It  disappears  rapidly  under  proper  quinine  treatment 
but  recovery  can  be  hastened  by  the  administration  of  iron  and 
arsenic. 

1.  Tertian  malaria  is  distinguished  by  a  chill  and  fever  occurring 
every  other  day,  the  patient  feeling  quite  well  on  fever-free  days. 
A  double  tertian  infection  occurs  not  infrequently,  giving  a  daily, 
or  quotidian,  chill  and  fever  with  no  free  day. 

2.  Quartan  fever,  which  is  relatively  rare,  gives  a  chill  and  fever 
every  third  day,  with  two  fever-free  days.    In  this  disease  also  there 
may  be  double   or   even  triple  infections,   giving  a   quotidian  or 
irregular  type  of  fever. 

3.  ^Estivo-autumnal   fever    (subtertian,    or    malignant   tertian) 
shows    an   irregular    temperature   curve,    the    cycle    varying   from 
twenty-four  to  forty-eight  hours.     By  some  authors  this  type   of 
malaria  is  divided  into  two  forms,  quotidian  and  tertian,  the  former 
giving  a  minute  ring,  the  latter  a  larger  one.    As  multiple  infections 
are  common  the  resulting  fever  curve  may  be  irregular  or  continuous 
and  the  chill  entirely  absent.    In  contrast  to  the  regular  intermit- 
tency  of  tertian  and  quartan  this  form  is  often  remittent,  the  tem- 
perature curve  never  dropping  to  normal. 

4.  Mixed  infections  with  any  two  of  the  above  fevers  are  often 
found  in  bad  malarial  regions  in  the  tropics. 

5.  Latent  malaria  is  also  not  infrequent,  in  which  the  patient, 
having  no  symptoms  of  the  disease,  consults  a  physician  for  some 
other  reason. 

In  a  region  where  the  disease  is  very  prevalent,  Bass*  has  shown 


*  C.  C.  Bass,  South.  Med.  Journ.,  1919,  12,  p.  460. 


SPOROZOA  1117 

that  almost  half  (45  per  cent)   the  infections  are  latent,  give  rise 
to  no  symptoms  and  have  negative  histories. 

6.  The  carrier  state  is  found  among  natives  or  persons  long 
resident  in  malarial  regions,  and,  aside  from  the  presence  of  a 
large  spleen  and  some  secondary  anemia,  may  present  no  symptoms. 
It  is  particularly  common  among  native  children,  tramps  and  vaga- 
bonds. It  is  not  uncommon  to  find  fifty  to  one  hundred  per  cent 
of  the  children  in  a  native  village  harboring  the  parasite. 


THE  DEVELOPMENT  OF  THE  HUMAN  MALARIAL  PARASITE 
IN  THE  MOSQUITO 

(Sexual  half  of  the  life  cycle,  Sporogonie) 

For  this  stage  to  be  successful  the  mosquito  must  bite  a  malarial 
patient  with  gametes  in  his  blood,  for  if  the  patient  be  one  in  the 
first  stage  of  the  disease,  with  only  schizonts  in  his  blood,  no  infec- 
tion of  the  mosquito  will  take  place,  since  the  schizonts  all  perish 
in  its  stomach.  On  the  contrary,  if  the  mosquito  takes  blood  from 
a  person  who  has  been  ill  with  malaria  for  some  time,  or  from  an 
apparently  healthy  carrier,  the  schizonts  die  as  usual,  but  the 
gametes  find  in  the  mosquito  stomach  for  the  first  time  conditions 
suitable  for  their  further  development. 

The  various  stages  may  be  studied  by  causing  suitable  species 
of  anophelin.es  to  bite  persons  with  many  gametes  in  their  blood, 
and  then  dissecting  the  stomach  and  observing  the  changes  which 
take  place  there.  The  development  is  visible  in  unstained  specimens 
with  high,  dry  lenses.  Since  there  is  no  essential  difference  in  the 
development  of  the  three  forms  of  malaria  in  the  mosquito,  they  will 
be  considered  together.  The  first  stage  has  already  been  described 
in  discussing  the  appearance  and  behavior  of  gametes  in  fresh  blood. 
In  the  mosquito  the  process  may  be  followed  further;  the  macro- 
gamete,  freed  from  its  enveloping  red  cell,  projects  a  little  mound 
on  its  surface,  and  this  apparently  attracts  the  microgametes  to 
its  neighborhood.  Into  this  microphyle  one,  but  never  more,  of  the 
flagella  penetrates,  following  which  the  mound  is  instantly  retracted. 
The  fertilized  macrogamete,  now  called  a  "zygote,"  soon  develops 
the  power  of  vermicular  motion,  then  being  called  an  ookinet,  and 
travels  to  the  wall  of  the  stomach,  and,  like  the  coccodia,  penetrates 
an  epithelial  cell  and  there  encysts,  making  the  oocyst.  This  grows 


HIS  PATHOGENIC   PROTOZOA 

rapidly  and  soon  escapes  from  its  host  cell  and  conies  to  lie  in  the 
outer  layers  of  the  stomach  wall,  and  as  it  grows  projects  into  the 
body  cavity  of  the  mosquito.  The  nucleus  divides  repeatedly,  always 
accompanied  by  some  of  the  cytoplasm,  forming  numerous  sporo- 
blasts,  and  these,  in  turn,  subdivide  into  innumerable  sporozoites; 
these  last  escape,  with  the  rupture  of  the  oocyst,  into  the  body 
cavity.  From  there  they  pass  to  all  parts  of  the  mosquito,  but 
especially,  perhaps  because  of  chemotaxis,  to  the  salivary  glands 
and  ducts,  and  when  next  the  mosquito  bites  a  warm-blooded  host 
the  sporozoites  enter  the  blood  stream  and  start  life  anew. 

Cultivation  of  the  Malarial  Parasites  in  Vitro.— Bass  and  Johns  in 
1911  announced  the  cultivation  of  a  few  generations  of  plasmodium 
vivax  in  vitro  under  strict  anaerobic  conditions.  Ten  c.c.  or  more  of 
blood  from  a  malarial  patient  is  defibrinated  and  distributed  in  small 
test  tubes  in  one  c.c.  quantities  and  to  it  is  added  one  per  cent  of  a 
fifty  per  cent  solution  of  glucose.  The  red  cells  settle  so  that  they 
are  covered  with  one-half  cm.  of  serum;  the  parasites  grow  in  a 
thin  layer  near  the  top  of  the  cell  mass;  beneath  this  they  die,  or 
are  phagocyted.  The  optimum  temperature  is  19°  to  40°  C. 

Bass  states  that  he  has  cultivated  all  three  species  of  plasmodia 
by  destroying  the  complement  by  heating  one-quarter  to  one-half 
hour  at  40°  C.  Under  strict  anaerobiasis  it  was  possible  to  transfer 
the  cultures  and  to  keep  them  alive  for  twenty  days. 

Transmission. — Transmission  is  solely  by  various  species  of 
anopheles  mosquitoes.  Although  there  are  fifty  or  more  recognized 
species,  only  sixteen  have  been  proved  malarial  carriers.  The  more 
important  are  Anopheles  quadrimaculatus  in  the  United  States, 
Anopheles  albimanus  in  the  American  tropics,  Anopheles  maculipennis 
in  Europe,  Anopheles  senensis  in  India,  and  Anopheles  costalis  in 
Africa. 

Description  of  the  Mosquito. — It  is  impractical  to  give  more  than 
a  hasty  description  of  mosquitoes,  and  the  reader  is  referred  to 
larger  works  on  the  subject  (Howard,  Dyer  and  Knab;  Theobald 
or  "Medical  Entomology,"  Patton  and  Oragg,  London,  Madras  and 
Calcutta,  1913).  Malaria  at  Home  and  Abroad,  S.  P.  James,  1920. 
It  may  be  noted,  however,  that  the  Culicidw,  or  mosquitoes,  belong 
to  the  Dipt  era,  or  two-winged  insects.  They  pass  through  four  distinct, 
stages  in  their  development,  the  egg,  larval,  pupal  and  adult  or  imago 
stage.  The  eggs  are  invariably  laid  in  water  where,  if  the  temperature 
is  warm,  they  hatch  in  one  to  four  days.  Anopheles  eggs  are  single 


SPOROZOA  1119 

and  oval,  supported  on  the  surface  of  the  water  by  ornamental  air 
cells;  those  of  culex  are  cemented  together  when  laid  in  raft-like 
masses. 

The  larva  are  aquatic  and  die  quickly  out  of  water.  They  are 
both  bottom  and  top  feeders,  eat  voraciously,  consuming  alga?  and 
other  vegetable  matter,  and  some  varieties  are  cannibalistic.  The 
larvae  are  provided  with  a  breathing  tube  or  respiratory  siphon  pro- 
jecting upward  from  the  dorsal  surface  at  the  caudal  end,  and  in 
breathing  this  is  thrust  upward  to  the  surface  of  the  water  and  the 
larva  hangs  suspended  from  the  surface  film.  In  the  anophelines  the 
breathing  tube  is  short  and  its  angle  with  the  body  is  such  that  the 
larva  lies  parallel  with  the  surface;  with  culex  and  other  genera  the 
body  lies  at  an  angle  with  the  surface.  The  larval  stage  lasts  about 
six  to  fourteen  days,  depending  upon  the  temperature  and  food  sup- 
ply, and  is  followed  by  the  pupal  stage,  during  which  no  feeding 
occurs;  the  pupa,  however,  needs  air  and  is  provided  with  a  short 
respiratory  tube  at  each  side  of  the  head.  The  habit  of  coming  to 
the  surface  of  the  water  to  obtain  air,  which  obtains  in  all  mosquitoes 
except  the  Mansonia,  gives  a  point  of  attack  in  combating  them,  since 
a  layer  of  mineral  oil  on  the  surface  of  the  water  occludes  the  respira- 
tory siphon  and  so  kills  them. 

The  adult,  or  imago,  emerges  from  the  pupa  when  the  latter  is  one 
to  three  days  old;  the  pupal  case  ruptures  along  its  dorsum  and  the 
emerging  imago  rests  on  the  floating  pupal  case  until  its  wings  are 
dry.  Since  this  is  a  critical  stage  in  its  life  history  and  demands 
quiet  water,  it  is  evident  that  the  least  wave  action  is  fatal  to  the 
mosquito. 

The  nature  of  the  breeding  place  is  characteristic,  to  a  certain 
extent,  of  each  genus  of  culicidce;  stegomyia  (cedes  calopus),  the  car- 
rier of  yellow  fever,  for  example,  is  strictly  domestic  and  breeds  in 
water  jars,  tin  cans,  old  beer  bottles  and  other  artificial  collections 
of  water,  ivyeomyia  breeds  exclusively  in  the  fluid  at  the  base  of  the 
leaves  of  air  plants  (bromeliads)  •  the  anophelines,  while  domestic  to 
the  extent  that  they  live  near  human  habitations,  require  natural 
collections  of  water  for  breeding  places,  such  as  sheltered  spots  along 
the  overgrown  banks  of  streams,  temporary  puddles  and  even  in 
water  in  the  footprints  of  man  and  animals. 

A  proper  classification  of  mosquitoes  requires  considerable  train- 
ing, but  the  following  points  will  suffice  to  separate  the  anophelines 
from  other  mosquitoes.  On  either  side  of  the  proboscis,  as  seen 


1120 


PATHOGENIC  PROTOZOA 


FIG.  161. — COMPARISON  OF  CULEX  (LEFT)  AND  ANOPHELES  (RIGHT).  Eggs;  larvae 
(note  position) ;  position  of  insects  at  rest;  wings;  heads  showing  antenna  and 
palpi.  (After  Kolle  and  Hetsch.  Jordan,  "General  Bacteriology,"  Saunders.) 


SPOROZOA  1121 

under  a  hand  lens,  are  two  pairs  of  organs;  next  to  the  proboscis 
are  the  palpi,  and  outside  of  these  the  antennae ;  the  latter  serve 
to  distinguish  the  sexes,  the  antennae  of  the  male  being  heavily 
ornamented  with  a  bushy,  hairy  investment  (plumose)  ;  the  female 
antennae,  on  the  contrary,  are  provided  with  relatively  few,  short 
hairs,  arranged  in  rings  at  the  joints  (pilose).  In  the  anophelines 
the  palpi  in  both  sexes  are  long,  at  least  as  long  as  the  probocis, 
while  in  all  other  mosquitoes  they  are  short  in  the  female  or  in 
both  sexes.  This  is  the  principal  differential  point.  The  wing  mark- 
ings are  of  some  help,  since  anopheline  wings  are  almost  always 
spotted.  Quite  characteristic  also  is  the  position  assumed  by  both 
genera  while  at  rest;  among  the  anophelines  the  head,  thorax  and 
abdomen  are  all  in  a  straight  line  and  the  insect  makes  an  angle 
with  the  surface  upon  which  it  rests;  while  the  culicidae  are  hump- 
backed, the  thorax  and  head  are  bent  on  the  abdomen  so  that  the 
latter  lies  parallel  to  the  surface.  By  these  characteristics  it  is 
easy  to  identify  a  mosquito  as  an  anopheline,  but  the  further  classifi- 
cation into  species  is  less  simple,  and  works  on  entomology  must 
be  consulted. 

The  life  history  of  the  anophelines  is  not  yet  completely  known ; 
they  fly  and  bite  at  dusk  and  dawn  and  during  the  night,  and  thus 
differ  from  stegomyia  and  most  other  culicidae,  which  are  day-time 
biters.  Their  breeding  places  have  already  been  described ;  of  special 
importance  are  the  temporary  collections  of  water  in  which  the 
larvae,  unhampered  by  their  natural  enemies,  quickly  reach  maturity 
in  large  numbers.  With  abundance  of  food  and  warm  weather,  the 
larva?  may  require  110  more  than  ten  days  to  complete  their  three 
molts.  The  female  alone  sucks  blood,  the  male  living  on  fruit  and 
vegetable  juices.  Egg-laying  does  not  take  place  until  after  a  meal 
of  blood,  and  it  is  possible  that  the  female  journeys  fairly  long 
distances  to  obtain  this  food,  and  that  the  first  flight,  from  breeding 
place  to  human  habitations,  may  be  longer  than  subsequent  flights. 
In  general  the  flight  is  short,  not  over  three  hundred  yards,  and 
Gorgas  found,  in  Panama,  that  a  clearing  of  that  width  about  houses 
gave  ample  protection. 

The  incubation  period  of  the  parasite  in  the  mosquito  is  about 
twelve  days,  after  which  the  insect  remains  a  carrier  during  the 
rest  of  its  life,  and  as  the  health  of  the  mosquito  is  unaffected,  this 
may  be  for  two  months  or  more. 


1122  PATHOGENIC   PROTOZOA 

Epidemiology. — Since  malaria  is  conveyed  solely  by  the  bite  of 
an  infected  anopheline,  the  epidemiology  is  comparatively  simple. 
In  practice,  nevertheless,  the  prevention  of  the  disease  is  extremely 
difficult;  but  it  is  the  same  for  all  forms  of  malaria.  It  must  be 
attacked  from  all  possible  angles  and  the  following  are  the  main 
points  to  be  observed : 

1.  Screened  houses  afford,  perhaps,  the  simplest  form  of  protec- 
tion, and  in  beginning  work  in  a  new,  badly  infected  place,  should 
be  the  first  thing  provided,  since  they  afford  a  place  of  security  in 
an  otherwise  dangerous  area,  where  the  workers  may  take  refuge 
until  the  situation  is  under  control.     This  method  alone  has  given 
magnificent  results  in  Italy  (Celli)  since  it  was  first  used  experi- 
mentally by  Sambon  and  Low  in  the  Roman  Campagna.  The  screens, 
to  be  durable,  must  be  of  bronze  and  not  iron,  and  of  a  fine  mesh 
(20  strands  to  the  inch),  and  should  .be  placed,  not  on  windows  and 
doors,  but  on  the  outside  of  porches  and  balconies ;  doorways  should 
have  screened  vestibules. 

In  default  of  metallic  house  screens,  bed  nets  may  be  used, 
although  they  are  not  very  satisfactory,  since  one  must  retire  at 
dusk  to  be  protected.  In  default  of  both  screens  and  bed  nets, 
something  may  be  accomplished,  temporarily,  by  daily  mosquito 
catching,  and  in  Panama  the  method  has  given  remarkable  results. 
A  native,  armed  with  a  small  acetylene  lantern  and  a  few  catching 
bottles,  soon  becomes  expert,  arid  can  capture  each  day  all  the 
mosquitoes  in  a  number  of  dwellings.  In  this  way  very  few  anophe- 
lines  escape  capture  long  enough  to  become  infective  for  man. 
Chloroform  catching  bottles  are  easily  prepared  by  packing  a  half 
ounce  of  small  rubber  bands,  cut  up  finely,  into  the  bottom,  and 
pouring  in  as  much  chloroform  as  the  rubber  will  absorb  and 
covering  it  over  with  dry  blotting  paper.  The  wide-mouthed  catch- 
ing bottle  is  uncorked  and  inverted  over  the  resting  mosquito,  which 
is  killed  by  the  chloroform.  A  convenient  trap  bottle  has  been 
described  by  La  Prince.  Below  the  cork  is  placed  a  funnel  trap, 
making  it  possible  to  pass  from  one  spot  to  another  without  waiting 
for  the  chloroform  to  act  upon  the  mosquito. 

2.  The  second  measure  is  the  prevention  of  mosquito  breeding; 
this  is  a  large  but  not  a  hopeless  undertaking,  if  carried  out  intel- 
ligently.    In  the  first  place,  it  is  to  be  remembered  that  compara- 
tively few  mosquitoes  are  disease  carriers,  and  that  measures  need 
be  directed  against  them  only.     It  was  first  shown,  for  example,  by 


SPOROZOA  1123 

Gorgas,  in  Havana,  that  stegomyia  could  be  practically  exterminated 
by  doing  away  with  water  in  artificial  containers  in  the  neighbor- 
hood of  dwellings;  this  so  reduced  their  numbers  that  when  a  case 
of  yellow  fever  was  introduced  into  a  community  the  disease  would 
not  spread.  In  the  same  way  malaria-carrying  mosquitoes  may  be 
fought,  without  regard  to  the  presence  of  non-disease  carriers.  An 
accurate  mosquito  survey  is  first  made,  both  by  examination  of  the 
catch  of  adults  and  by  a  hunt  for  larvae  in  collections  of  water 
throughout  the  district.  A  puddle  or  stream  is  examined  by  dipping 
with  a  white  saucer  or  a  long-handled  dipper,  along  the  margins; 
after  a  little  practice  it  is  possible  to  obtain  any  larvae  which  may 
be  present,  and  to  decide  by  their  appearance  and  behavoir  whether 
they  are  anophelines.  It  is,  of  course,  unnecessary  to  waste  time 
and  money  destroying  collections  of  water  free  from  anophelines. 
When  the  breeding  places  have  been  located,  they  may  be  destroyed 
by  the  use  of  oil  or  larvacide,  by  draining  or  by  filling. 

The  use  of  oil  and  larvacides,  while  usually  a  temporary  measure, 
is,  nevertheless,  of  great  importance ;  any  light  fuel  oil  may  be  used 
by  spraying,  by  mopping  the  sides  of  ditches  or  margins  of  ponds, 
or  by  a  drip  barrel  at  the  head  of  a  water  course.  Since  in  the 
malarial  season  the  mosquito  develops  rapidly  the  oil  must  be  applied 
regularly  once  a  week.  It  is  better  in  the  long  run  to  begin  in  the 
center  of  the  settlement,  with  permanent  improvements,  working 
outwards  as  money  becomes  available,  and  not  to  depend  on  oiling 
except  as  a  temporary  measure. 

Wherever  possible,  the  swampy  areas  and  pools  must  be  drained, 
and  for  this  purpose  both  agricultural  tile  and  open  ditches  may  be 
used;  in  the  tropics  it  is  necessary  to  line  the  latter  with  concrete 
to  prevent  overgrowth  by  rank  vegetation  and  to  protect  the  banks 
against  caving.  Where,  because  of  the  lay  of  the  land,  drainage  is 
impracticable  the  area  may  be  filled,  or  sometimes  flooded,  or 
irrigated  with  sea  water,  in  which  most  anophelines  do  not  survive. 

3.  The  infection  is  kept  alive  in  a  community  by  human  carriers, 
and  these  are  especially  common  among  natives  and  the  poor  and 
ignorant,  and  especially  among  the  native  children.  Newcomers 
should  not  live  within  five  hundred  yards  of  the  dwellings  of  these 
classes,  as  infected  anophelines  can  easily  travel  shorter  distances. 
Only  when  it  is  possible  to  protect  the  natives  also  by  these  measures 
is  it  safe  to  live  among  them;  and  that  this  is  possible  has  been 
shown  many  times,  particularly  in  Panama. 


1124  PATHOGENIC  PROTOZOA 

4.  The  proper  treatment  and  cure  of  all  cases  will  not  only 
prevent  relapses  and  the  carrier  state  (malarial  cachexia),  but  is 
one  of  the  most  important  means  of  exterminating  the  disease,  since 
every  neglected  case  becomes  a  focus  for  new  mosquito  and  in  conse- 
quence new  human  infections.  It  is  just  as  important  a  part  of  the 
prevention  of  the  disease  as  any  other  single  measure,  and  in  general 
has  been  given  too  little  consideration.  Between  50  and  90  per  cent 
of  all  malarial  attacks  are  relapses  and  not  new  infections,  and 
these  cases  can  be  cured  and  the  sick  rates  be  reduced  accordingly 
by  giving  proper  and  adequate  treatment  during  the  acute  attack 
and  during  convalescence.  This  means,  in  effect,  that  the  education 
of  the  physicians  and  of  the  community  in  general  must  be  under- 
taken by  the  health  authorities.  It  is  not  sufficient  merely  to  obtain 
the  support  of  the  physicians  in  any  given  locality,  for  perhaps  not 
more  than  25  per  cent  of  all  cases  of  malaria  ever  consult  a  physician. 
Many  now  buy  various  advertised  remedies  most  of  which  are  of 
little  value,  but  so  far  as  they  are  curative,  the  good  effect  is  without 
doubt  due  to  the  quinine  which  they  contain.  The  general  public 
can  be  influenced  to  buy  and  take  quinine  in  adequate  doses  over  a 
considerable  period  by  the  educational  division  of  the  Health  Depart- 
ment. Bass  in  Mississippi  has  obtained  excellent  results  by  using 
and  advocating  the  use  of  a  standard  treatment  of  ten  grains  a  day, 
after  the  acute  attack  is  past,  for  a  period  of  eight  weeks.  While 
this  will  not  cure  all  cases,  it  is  a  great  improvement  upon  the 
system  of  treatment  commonly  used.  To  provide  as  far  as  possible 
for  the  continuation  of  the  treatment  for  the  regular  period  of 
eight  weeks,  the  standard  package  sold  and  recommended  by  the 
local  health  authorities  contains  enough  quinine  for  the  complete 
treatment,  and  it  is  impossible  to  buy  a  part  of  it  at  the  official 
price.  The  immediate  effect  upon  the  morbidity  and  mortality  rates 
of  adequate  treatment  of  malarial  attacks  is  immediate  and  very 
satisfactory.  It  is  not  so  much  a  question  of  educating  the  public 
to  take  medicine  for  their  illness,  they  already  do  that  in  some 
form  or  other,  as  it  is  to  educate  them  to  take  quinine  in  proper 
doses.  The  influence  upon  the  amount  of  malaria  in  a  community 
of  proper  treatment  of  all  cases  is  difficult  to  estimate,  because  of 
the  shifting  of  population  in  malarious  regions,  but  it  promises  to 
diminish  the  number  of  carriers  and  correspondingly  the  number 
of  foci  of  infection. 


SPOROZOA  1125 

5.  Quinine  prophylaxis  is  an  unsatisfactory  measure  which  must 
be  used  by  travelers,  explorers  and  troops.     There  is  no  method  of 
using  quinine  which  will  entirely  prevent  malaria  when  the  chances 
for  infection  are  many;  the  following'  methods  have  all  been  used: 
(1)  The  so-called  gram  prophylaxis,  in  which  one  gram  of  quinine 
is  taken  intermittently  every  tenth  day  as  the  minimum  to  every 
fourth  day  as  the  maximum.     The  gram  may  be  taken  in  a  single 
dose  at  bed-time,  or  in  four  divided  doses  during  the  day-time.    (2) 
The  double  gram  prophylaxis,  in  which  the  dose  is  taken  on  two 
successive  days,  as,  for  example,  on  the  tenth  and  eleventh,  or  on 
the  fifth  and  sixth.     (3)  The  half  gram  prophylaxis,  as  proposed  by 
A.  Plehn,  was  0.5  gram  every  fifth  day;  experience  has  shown  that 
it  is  of  little  value.     (4)  A  daily  dose  of  0.4  to  0.8  gram  gives  better 
results  than  any  other  method,  since  the  patient  suffers  less  from 
cinchonism  than  when  larger  doses  are  taken  intermittently,  and 
takes  his  quinine  more  faithfully.    The  size  of  the  dose  depends  on 
the  form  of  fever  present  and  the  number  of  chances  for  infection; 
0.4  gram  will  often  protect  against  tertian  and  quartan,  while  even 
0.8   may   fail   to   prevent   aestivo-autumnal.      Latent    infections   and 
relapses   among  "prophylacticers"   are   common   and   black   water 
fever  is  not  an  infrequent  sequel. 

It  is  well  to  remember  that  quinine  is  rapidly  absorbed  from 
the  intestinal  tract  and  also  as  rapidly  excreted,  and  that  so  far 
as  possible  the  doses  should  be  properly  timed. 

It  is  probable  that  quinine  is  already  in  the  blood  within  half 
an  hour  of  the  time  it  is  taken,  and  that  the  whole  dose  is  absorbed 
within  six  hours  and  that  most  of  it  has  already  been  excreted  in 
the  same  period.  To  provide  quinine  in  the  blood  in  sufficient  con- 
centration to  be  effective  in  killing  the  sporozoites  injected  by  the 
mosquito,  it  is  most  reasonable  to  take  one  dose  of  five  grains  an 
hour  before  sunset,  and  a  second  dose,  if  one  is  compelled  to  be 
out,  just  before  midnight.  Ten  grains  taken  in  this  way  will  provide 
against  infection  at  dusk  and  at  dawn,  the  two  principal  periods 
during  which  the  anophelines  bite,  better  than  one  dose  at  bed  time, 
of  the  same  size. 

6.  Personal  prophylaxis  by  means  of  head  nets,  gloves,  suitable 
clothing,  and  the  use  of  essential  oils,  such  as  citronella,  on  exposed 
parts  of  the  body,  is  helpful  in  emergencies. 

7.  The  protection  of  human  beings  by  means  of  animal  barriers 
has  been  recommended  by  several  writers.     The  subject  has  been 


1126  PATHOGENIC   PROTOZOA 

reviewed  by  Roubaud,*  who  has  been  much  impressed  by  the  prac- 
tical disappearance  of  malaria  from  France  without  a  corresponding 
diminution  of  anophelines  capable  of  acting  as  carriers.  In  La 
Vendee,  a  Department  of  France,  are  regions  where  there  are  many 
swamps  and  few  cattle  or  domestic  animals  and  it  is  in  such  regions 
that  human  malaria  remains;  in  other  regions,  abundantly  supplied 
with  domestic  animals  the  disease  has  practically  disappeared  al- 
though anophelines  still  may  be  found  in  the  animal  shelters.  He 
believes  that  the  insects  prefer  the  blood  of  cattle  and  horses  to  that 
of  human  beings,  and  that  a  sufficient  number  of  animals  will  protect 
the  human  inhabitants  against  infection  in  an  otherwise  malarious 
locality.  The  method  is  of  sufficient  importance  to  merit  further 
study  and  trial. 

In  conclusion,  it  may  be  affirmed  that  any  district,  no  matter 
how  notorious,  may  be  freed  from  malaria,  if  necessity  demand  it 
and  money  be  forthcoming,  by  means  of  the  above  measures. 

Pathology. — The  pathological  features  of  the  disease  are  quite 
definite ;  at  autopsy  there  is  evidence  of  some  secondary  anemia,  due 
to  the  destruction  of  enormous  numbers  of  erythrocytes ;  the  hemo- 
zoin,  in  well-marked  cases,  accumulates  in  the  viscera  until  they 
are  chocolate  or  slate  colored;  the  spleen  is  enlarged  and  friable, 
and  the  liver  and  kidneys  may  show  cloudy  swelling.  In  smears 
prepared  from  the  spleen,  liver,  kidneys,  brain  and  bone  marrow, 
parasites  and  hemozoin  will  be  found,  although  the  former  may  not 
be  numerous.  The  distribution  of  the  parasites  is  usually  unequal, 
but  they  are  often  present  in  the  spleen  and  brain  in  greatest 
numbers. 

The  origin  of  the  pigment  has  already  been  described;  it  is 
phagocyted  and  accumulates  in  the  viscera,  but  after  a  time  dis- 
appears in  some  unknown  way ;  its  presence,  therefore,  is  an  indica- 
tion of  malaria  in  recent  years.  It  must  be  distinguished  from 
hemosiderin,  a  yellowish  pigment  found  in  the  viscera  after  extensive 
destruction  of  red  blood  cells.  Hemozoin  is  soluble  in  alkalis  and 
insoluble  in  acids,  water,  chloroform,  alcohol  and  ether,  while  hemo- 
siderin is  insoluble  in  acids  and  alkalis  but  soluble  in  alcohol.  Both 
contain  iron,  yet  the  former  (hemozoin)  does  not  give  a  Berlin  blue 
reaction,  while  it  is  present  with  the  latter. 

*  Roubaud,  E.,  Les  Conditions  <le  nutrition  des  anopheles  en  France  et  le  role 
du  Betail  dans  la  prophylaxie  du  paludisme.  Ann.  Inst.  Pasteur.,  Paris,  1920, 
34,  181. 


SPOROZOA  1127 

The  leucocytes,  although  increased  during  paroxysm,  are  soon 
diminished  so  that  leucopenia  is  characteristic  of  the  disease ;  in 
addition,  there  is  a  relative  increase  in  the  mononuclear  leucocytes. 
The  loss  of  hemoglobin  is  very  great,  yet  is  quickly  recovered  from 
during  convalescence. 

In  fatal  cases  of  aestivo-autumnal  fever  a  striking  feature  is  the 
presence  of  innumerable  infected  red  cells  in  the  capillaries  of  the 
brain  or  abdominal  viscera.  A  smear  from  a  pigmented  brain  may 
show  a  capillary  thrombus  made  up  of  infected  erythrocytes,  most  of 
them  showing  the  sporulatmg  stage.  In  deaths  after  repeated  ma- 
larial attacks  the  kidneys  will  show,  in  addition  to  the  presence  of 
many  parasites,  a  marked  chronic  diffuse  nephritis,  one  of  the  most 
important  sequelae  of  the  disease. 

Immunity. — The  disease  is  strictly  confined  to  human  beings,  as 
none  of  the  lower  animals  are  susceptible ;  there  appears  to  be  some 
racial  and  acquired  immunity,  although  it  is  incomplete;  in  native 
settlements  the  number  of  children  showing  parasites  in  the  blood  is 
much  greater  than  the  number  of  adults,  yet  the  latter  are  not  abso- 
lutely immune;  one  attack  certainly  gives  no  protection  against  a 
new  infection.  The  evidence  from  different  regions  is  quite  con- 
flicting in  regard  to  immunity  and  has  been  most  plausibly  explained 
in  this  way ;  where  the  disease  prevails  throughout  the  year  there  is 
a  constant  reinfection  and  under  this  stimulus  the  body  is  able  to 
keep  up  an  immunity  strong  enough  to  kill  off  the  parasites  before 
any  symptoms  arise;  in  other  regions,  however,  where  malaria  is  a 
seasonal  disease,  the  constant  stimulus  is  lacking  and  there  is  less 
evidence  of  immunity.  It  has  also  been  noted  that  the  three  varieties 
of  the  disease  are  distinct,  and  that  no  immunity  against  the  whole 
group  is  obtained  by  an  infection  with  one  species. 

Since  many  mild  cases  recover  without  treatment,  it  is  apparent 
that  some  little  immunity  is  produced  by  an  infection,  yet  it  is  tem- 
porary and  does  not  protect  against  repeated  relapses.  Before  the 
days  of  radical  treatment  with  quinine,  relapses  were  the  rule  and 
were  considered  an  essential  feature  of  the  disease  (Manneberg). 

Clinical  experience  teaches  that  relapses  occur  when  treatment 
has  been  insufficient,  and  especially  after  fatigue,  getting  wet  and 
catching  cold,  or  after  over-heating  in  the  tropical  sun,  and  par- 
ticularly after  a  sea  voyage  or  a  long  journey.  They  may  continue 
to  occur  in  a  region  free  from  malaria,  for  about  three  years ;  in  a 
malarial  region  it  is  difficult  to  differentiate  between  relapses  and 


1128  PATHOGENIC  PROTOZOA 

reinfections.  They  are  most  frequent  after  quartan,  then  tertian, 
and  least  following  asstivo-autumnal. 

The  cause  of  the  relapse  is  still  a  matter  of  discussion ;  Schaudinn 
explained  it  as  due  to  parthenogenesis,  the  macrogamete  changing  to 
a  schizont  after  expelling  a  part  of  its  nucleus  and  cytoplasm  and  so 
starting  a  new  cycle  of  asexual  parasites.  Craig  has  suggested  an 
intra-corpuscular  conjugation  as  the  beginning  of  a  new  crop  of 
parasites.  It  is  possible  that  the  old  theory,  that  parasites  survive 
for  long  periods  in  the  viscera,  is  correct  and  that  the  relapse  is 
brought  about  by  any  condition  which  temporarily  reduces  the  con- 
valescent's immunity,  such  as  fatigue,  etc.  H.  C.  Clark,  in  Panama, 
has  examined  a  large  series  of  placentas  from  apparently  healthy 
women  and  found  that  not  infrequently  they  contained  plasmodia  in 
large  numbers,  even  in  the  absence  of  recent  malarial  attacks.  There 
was,  evidently,  sufficient  immunity  to  hold  the  parasites  in  check 
under  ordinary  circumstances. 

Under  ordinary  conditions  the  disease  prevails  more  extensively 
and  in  a  more  severe  form  among  recent  arrivals  in  a  district  than 
among  the  older  residents ;  while  this  is  no  doubt  partly  due  to  an 
acquired  immunity,  it  is  not  improbable  that  education  in  the  proper 
methods  of  treatment  of  the  acute  disease,  to  prevent  relapses;  in 
the  avoidance  of  notoriously  dangerous  areas,  and  in  the  selection 
for  favorable  sites  for  dwelling,  and  the  protection  of  the  individual 
by  screens,  are  of  equal  importance  in  protecting  the  older  residents. 

A  specially  severe  form  of  malaria  has  been  called  "campaign 
malaria,"  which  develops  among  troops  in  endemic  areas.  In  such 
cases  the  housing,  and  food  conditions  are  usually  poor,  the  men 
are  engaged  in  hard  and  dangerous  work  at  all  hours  of  the  day 
and  night,  with  the  result  that  they  are  repeatedly  Htten  by 
anophelines,  and  their  blood  shows  a  multiple  infection. 


BLACK  WATER  FEVER 

This  condition  follows  malaria  and  occurs  only  in  malarial  dis- 
tricts, and  by  most  authorities  is  believed  to  be  a  sequel  of  the 
disease,  due  to  some  unknown  factor.  It  is  characterized  by  the 
passage  of  urine  containing  hemoglobin,  albumin  and  casts.  In 
color  the  urine  varies  from  a  pale  red  to  a  black.  Castellani  and 
Chalmers,  however,  separate  it  into  three  forms,  symptomatic,  toxic 


SPOROZOA  1129 

and  specific  hemoglobinuria,  examples  of  which  are,  respectively, 
malarial  and  quinine,  hemoglobinuria  and  black  water  fever.  In 
none  of  these,  however,  is  the  etiology  clear;  the  disease  does  not 
occur  independently  of  malaria,  and  an  attack  may  be  precipitated 
or  aggravated  by  quinine.  Leishman  has  described  certain  cell 
inclusions,  possibly  chlamydozoa,  as  the  cause.  The  etiology  is,  at 
the  present  time,  far  from  clear.  Malarial  parasites  may  be  present 
in  the  blood  up  to  time  of  the  appearance  of  the  hemaglobinuria, 
but  then  they  rapidly  disappear  since  it  is  the  infected  parasites 
which  first  undergo  destruction.  To  determine  the  presence  of 
hemaglobin  with  certainty  the  urine  should  be  examined  with  a 
spectroscope;  small  models  are  made  which  are  suitable.  The 
hemaglobin  in  the  blood  falls  rapidly,  to  twenty-five  per  cent  or 
even  less ;  the  number  of  red  cells  is  also  diminished,  often  to  three 
million  per  cubic  millimeter  or  and  in  severe  cases  to  a  much  lower 
figure. 

In  these  cases  quinine  must  be  stopped  until  the  hemaglobinuric 
attack  is  over,  when  the  parasites  reappear  and  the  malaria  must 
again  be  treated.  In  the  mean  time  the  patient  should  be  kept  in 
bed  and  be  given  the  best  possible  care  and  nursing. 


TREATMENT  OF  MALARIA 

In  quinine  we  have  a  true  chemical  specific  for  malaria,  and  when 
given  early  enough  and  in  sufficient  doses,  will  cure  the  disease  with 
certainty.  It  is  usually  given  in  the  form  of  the  sulphate  or  dihydro- 
chlorate,  preferably  in  solution,  but  may  be  administered  in  freshly 
prepared  capsules;  pills  and  tablets,  while  convenient,  are  unsatis- 
factory because  of  their  relative  insolubility.  It  acts  vigorously  on 
the  merozoites  and  young  trophozoites,  but  has  almost  no  direct 
effect  upon  the  gametes.  The  size  of  the  dose  depends  on  the  form 
of  the  fever  and  its  severity.  In  ordinary  cases  five  grains  three 
times  a  day  is  sufficient,  while  in  severe  infections  not  less  than 
thirty  grains  a  day  must  be  given;  the  best  time  is  immediately 
after  meals,  without  regard  to  the  time  of  the  chill.  To  prevent 
relapses,  the  treatment  of  the  original  infection  must  be  thorough, 
and  the  patient  should  be  kept  in  bed,  upon  a  light  diet  and  attention 
paid  to  the  condition  of  the  bowels.  During  and  after  convalescence 
the  treatment  must  be  continued  for  three  months,  though  the  daily 


1130  PATHOGENS   PROTOZOA 

dose  may  be  decreased  gradually,  beginning  a  few  days  after  the 
subsidence  of  the  fever.  In  exceptional  cases  a  relapse  will  occur 
while  the  patient  is  still  taking  massive  doses  of  quinine,  and  by 
some  this  lias  been  looked  upon  as  an  evidence  of  immunity  of  the 
parasite  to  the  drug,  but  it  is  possible  that  it  is  merely  due  to  non- 
absorption  of  the  quinine,  and  carminatives  should  be  added  to  the 
dose  to  assist  in  its  absorption. 

The  addition  of  pepsin  to  the  quinine  will  reduce  the  unpleasant 
effects  of  the  drug.  To  ninety  parts  of  quinine  add  6  parts  of 
lactose  and  4  parts  of  pepsin. 

The  proportionate  dose  required  for  children  is  greater  than 
indicated  by  Cowling's  rule.  Bass  finds  that  for  children  of  fifteen 
or  over,  the  full  adult  dose  is  necessary.  From  eleven  to  fourteen 
0.8  of  the  adult  dose,  from  eight  to  ten  0.6,  from  five  to  seven  0.4, 
for  three  and  four  year  old  children  0.3,  two  year  old  0.2,  one 
year  old  0.1  of  the  adult  dose  is  necessary.* 

In  selected  cases  quinine  is  occasionally  given  subcutaneously  or 
intravenously  although  the  method  is  not  free  from  the  risk  of 
abscesses  in  the  subcutaneous  tissue,  or  death  after  intravenous 
use. 

For  intravenous  use  a  freshly  sterilized  solution  consisting  of 
12  to  15  grains  of  bihydrochlorid  of  quinine  dissolved  in  10  to  20 
c.c.  of  normal  salt  solution  can  be  injected  into  a  vein  at  the  rate  of 
half  a  c.c.  per  minute.  For  subcutaneous  use  the  same  quantity  of 
quinine  may  be  used  but  it  must  be  dissolved  in  a  few  cubic  centi- 
meters of  fluid.  The  injection  must  be  given  into  the  loose  arcolar 
tissue  but  even  then  it  is  often  painful  and  apt  to  be  followed  by 
necrosis  and  abscess. 

Treatment  with  arsphenamine  and  related  preparations. 

It  has  been  shown  that  the  administration  of  half  doses  of  the 
606  group  of  remedies  can  be  given  to  selected  cases  of  severe 
malaria  in  addition  to  the  usual  treatment  with  quinine,  with  benefit ; 
for  chronic  resistant  infections  and  for  malarial  cachexia  three  or 
four  injections  at  weekly  intervals  are  recommended.  The  tonic 
effect  of  these  drugs  is  quite  marked  and  the  duration  of  illness  is 
shortened  by  the  combined  treatment. 


*  Pratt- Johnson,  ,/.,  Gilchrist,  Kenneth,  and  Hay-Michel.  On  the  Action  of 
Certain  Special  Preparations  on  Malarial  Parasites  and  their  Employment  in  the 
Treatment  of  Malaria. 


SPOROZOA  1131 

The  gametocytes,  when  once  formed  resist  all  forms  of  treat- 
ment, but  under  energetic  quinine  medication  the  crescents  usually 
cease  to  be  produced  and  disappear  from  the  circulating  blood  in 
about  fifteen  days. 

OTHER  MALARIAL  PARASITES 

From  time  to  time  additional  malarial  parasites  have  been  de- 
scribed, only  two  of  which  are  of  any  importance  at  present:  Plas- 
m-odium vivax,  variety  minuta,  and  Plasmodium  tenue.  The  former 
was  described  by  Ahmed  Emin  in  1914.  It  resembles  the  usual  ter- 
tian in  general,  but  differs  in  the  following  points;  it  is  smaller,  the 
infected  erythrocyte  is  not  enlarged,  and  the  number  of  merozoites  is 
small  (four  to  ten).  The  pigment  is  fine  and  motility  is  not  marked. 
Multiple  infection  of  the  erythrocytes  is  not  uncommon.  Craig3  de- 
scribed a  similar  parasite  in  1900,  and  he  suggests  that  the  parasite 
has  been  confounded  with  Plasmodium  malarias. 

In  1914  Stephens4  described  an  organism  from  one  slide,  which  he 
calls  Plasmodium  tenue.  It  is  said  to  be  deficient  in  pigment,  mark- 
edly motile,  and  rich  in  chromatin.  Since  the  parasite  was  described 
from  one  slide,  it  is  very  doubtful  if  it  can  at  present  be  accepted  as 
a  valid  species. 

Piroplasmidse  (Franca). — This  is  a  provisional  family  belong  to 
the  hemosporidia,  the  type  of  which  is  Babesia  bigeminum,  Smith 
and  Kilborne  (pirosoma,  piroplasma),  the  cause  of  Southern  cattle 
fever. 

The  parasite  was  first  described  by  Smith  and  Kilborne  in  1889, 
and  correctly  placed  by  them  among  the  protozoa ;  they  also  demon- 
strated its  transmission  by  the  cattle  tick,  and  this  achievement 
marks  the  beginning  of  medical  protozoology.  To  the  original  para- 
site, the  cause  of  Texas  fever,  has,  in  course  of  time,  been  added 
other  forms  until  now  we  have  a  family  consisting  of  Babesia 
bigeminum,  bovis,  cam's,  equi,  ovis,  mutans,  quadrigeminum,  and  a 
closely  related  parasite,  Theileria  parva. 

Morphology. — The  parasites  are  pear-shaped,  round,  oval,  or  ame- 
boid, inhabiting  mammalian  red  blood  cells,  which  they  destroy  but 
without  producing  pigment.  In  entile,  sheep,  horses  and  dogs,  the 


3  Craig,  Jour.   Parasitol.,   Urbana,   Til.,    1914,   1,   88. 

*  Stephens,  Proc.  Roy.  Soc.,  Loncl.,  Series  B,  87,  p.  375. 


1132  PATHOGENIC  PROTOZOA 

freed  hemoglobin  is  excreted  by  the  kidneys,  producing  the  disease 
variously  known  as  red  water  fever,  Texas  or  Southern  cattle  fever, 
tick  fever,  bovine  malaria,  hemoglobinuria,  and  others.  The  parasite 
is  small,  two  to  four  microns  long  and  one  to  two  wide,  and  character- 
istically occurs  in  pairs,  the  narrow  ends  being  united.  When  the 
parasite  is  mature  the  two  daughter  cells  separate  and  when  liberated 
by  the  degenerated  erythrocyte  attack  new  red  cells.  The  pear-shaped 
babesia  enters  a  new  cell  by  its  broad  end,  becomes  rounded  or  ring- 
like,  then  ameboid  in  form,  and  finally  the  nucleus  sends  out  a  bud; 
this  divides  into  two  by  forking,  and  as  the  nuclear  matter  continues 


FIG.  162. — BABESIA   BIGEMINUM.     (Army    Med.    School   Collection, 
Washington,  D.  C.) 

to  grow  each  portion  becomes  surrounded  by  cytoplasm  and  ultimately 
the  two  new  daughter  cells  separate  from  one  another.  Multiple 
infections  of  single  red  cells  are  common,  as  many  as  sixteen  pairs 
having  been  seen  in  a  single  cell. 

Good  preparations  for  clinical  work  are  obtained  by  some  one  of 
the  Romanowski  stains,  but  the  finer  details  of  the  nucleus  and  cell 
division  can  only  be  studied  after  iron  hemotoxylin  staining. 

The  parasites,  with  the  exception  of  T'heileria  parva,  do  not  disap- 
pear completely  from  the  blood  after  the  animal  recovers  from  its 
illness,  but  remain  indefinitely  in  the  circulation,  and  their  continued 
presence  and  virulence  may  be  demonstrated  by  the  inoculation  and 
non-immune  animals  with  blood  from  an  animal  which  has  recovered. 


SPOROZOA  1133 

Transmission. — Transmission  from  host  to  host  is  by  means  of 
ticks.  Margaropus  annulatus  carries  Southern  cattle  fever  in  the 
United  States,  but  other  ticks  are  carriers  in  South  America  and  the 
West  Indies.  In  general,  each  species  of  babesia  is  specific  for  a  par- 
ticular animal,  and  each  is  carried  by  a  separate  group  of  ticks. 

Clinical  Observations. — Clinical  observations  have  shown  an  in- 
cubation period  of  about  fourteen  days;  the  onset  is  with  fever  and 
the  cattle  look  weak  and  ill,  neither  eat  nor  chew  the  cud,  but  stand 
with  sunken  head  and  relaxed  ears.  A  bloody  diarrhea  sets  in  early 
and  the  urine,  which  is  small  in  amount,  is  deep  red  in  color  and 
contains  much  albumin.  The  blood  shows  few  parasites  at  first,  but 
they  soon  increase  and  the  number  of  red  cells  falls  rapidly.  The 
mortality  varies  in  different  epidemics  from  five  to  sixty  per  cent, 
but  has  been  as  high  as  ninety  in  some  herds.  Young  cattle,  under  a 
year,  have  a  mild  form  of  the  disease,  and  remain  thereafter  immune. 

The  treatment,  once  the  disease  has  appeared,  is  unsatisfactory, 
since  we  have  no  specific,  but  much  may  be  done  by  prophylaxis. 
Quarantine  of  cattle  from  infected  regions  is  absolutely  necessary  to 
prevent  the  spread  of  the  disease,  since  all  immune  animals  are  also 
carriers.  Young  animals,  between  nine  and  twelve  months  of  age, 
may  be  inoculated  with  the  blood  of  those  which  have  recovered,  five 
to  ten  c.c.  being  given  in  a  single  dose ;  the  disease  lasts  two  to  three 
days,  and  the  mortality  is  not  great,  as  eighty  to  ninety  per  cent  of  the 
inoculated  animals  recover  and  remain  immune.  Inoculation,  how- 
ever, is  a  measure  of  doubtful  value  and  some  means  of  tick  eradi- 
cation should  be  used  if  the  disease  is  to  be  permanently  stamped 
out. 

Life  History  of  the  Tick  (Margaropus  annulatus)5 

The  tick's  life  is  divided  into  two  stages,  one  part  passed  on  cattle 
and  another  part  passed  on  the  ground.  The  mature  female,  as 
found  on  cattle,  is  about  half  an  inch  in  length,  plump  and  olive  green. 
When  fully  engorged  with  blood  from  its  host,  it  drops  to  the  ground, 
seeks  a  sequestered  hiding  place  and  if  it  escapes  birds,  ants  and 
other  enemies,  begins  after  a  few  days  of  warm  weather  to  lay  eggs. 
These  are  small,  elliptical,  at  first  light,  later  dark  brown,  and  are 
cemented  together  in  irregular  masses  by  a  sticky  secretion;  in  num- 
bers they  vary  from  a  few  hundred  to  more  than  five  thousand  for 

"Farmers'  Bull.  No.  498,  IT.  S.  Dept.  of  Agri. 


1134 


PATHOGENIC   PROTOZOA 


each  female.  The  female  tick  dies  in  a  few  days  after  the  egg-laying 
has  been  completed.  The  eggs  soon  hatch  (after  nineteen  days  in 
summer  to  one  hundred  and  eighty-eight  in  winter)  and  a  small,  oval, 
six-legged  larva  or  seed  tick  appears,  and  promptly  climbs  up  on  the 
nearest  vegetation,  grass,  weeds  or  bushes  to  lie  in  wait  for  a  warm- 
blooded host.  Although  while  on  vegetation  seed  ticks  do  not  take 
food  nor  grow,  their  endurance  is  great,  and  during  the  colder  parts 
of  the  year  they  may  live  for  eight  months.  The  next  stage  begins 
after  the  seed  tick  has  found  a  host,  when  it  sucks  blood,  increasing 
in  size  and  soon  (five  to  twelve  days)  molts  and  a  new  form,  the  eight- 


FIG.  163. — THE  TEXAS  FEVER  TICK  (Margaropus  annulatus). 
live  Medicine  and  Hygiene.") 


(Rosenau,  "Preveri- 


legged  nymph,  appears.  In  five  to  eleven  days  more  a  second  molt 
occurs  and  the  tick  is  then  sexually  mature  and  males  can  be  dis- 
tinguished from  females.  The  female  does  not  move  about  on  the 
animal,  but  the  male  seeks  her  out,  and  after  fertilization  growth  goes 
on  rapidly  until  the  engorged  female  drops  to  the  ground.  To  sum- 
marize :  On  the  ground  is  the  engorged  female,  the  eggs  and  seed  ticks ; 
on  the  animal  is  the  seed  tick,  the  nymph,  the  sexually  mature  adult 
and  finally  the  engorged  female.  The  infected  female  tick  transmits 
the  babesia  to  the  larvas  through  the  eggs,  but  does  not  herself  bit£ 
nor  convey  the  disease  directly  to  another  animal. 

It  is  evident  that  the  tick  may  be  attacked  in  the  pasture  or  on 
the  cattle.  Pasture  rotation  is  one  of  the  methods  recommended  by 
the  Agricultural  Department,  and  its  rests  upon  the  fact  that  all  the 
ticks  will  die  from  starvation  in  an  unused  pasture  in  from  six  to 
twelve  months,  varying  with  the  climate,  the  shorter  period  holding 
true  for  warmer  localities.  By  changing  pastures  a  farm  may  be  freed 


SPOROZOA  1135 

of  ticks  in  four  and  a  half  or  eight  months,  depending  on  the  plan 
followed.  For  economic  reasons,  lack  of  sufficient  pasture  land,  etc., 
this  plan  has  not  been  widely  adopted.  A  second  plan,  that  of  dipping, 
has  been  more  successful.  The  cattle  are  driven  through  a  large  dip- 
ping vat  at  intervals  of  two  weeks  (never  more  than  three  must 
elapse)  until  they  and  the  pasture  are  free  from  ticks.  The  fluid  in 
the  dipping  vats  is  an  alkaline  solution  of  arsenic;  oil  dips  are  little 
used  at  present.  Arsenical  dips  are  cheap,  easily  prepared  and  effi- 
cacious, two  or  at  most  three  dippings,  at  ten-day  intervals,  being  suf- 
ficient to  free  heavily  infected  cattle,  and  if  they  can  then  be  put  on 
tick-free  pastures  the  problem  is  solved.  If  no  tick-free  pastures  are 
available,  dipping  must  be  continued,  as  above,  until  the  animals 
remain  permanently  free. 

The  prevention  of  tick-borne  disease  is  to  be  solved,  therefore,  by 
tick  eradication,  and  experience  in  the  Southern  states  has  shown  this 
to  be  a  practical  measure. 


SUB-CLASS— NEOSPORIDA 

SARCOSPORIDIA 

These  organisms  belong  to  the  sub-class  Neosporidia  of  the  Sporo- 
zoa,  because  the  spore  formation  commences  before  the  completion 
of  growth.  They  have  been  known  since  1843,  when  Miescher  dis- 
covered ''tubes"  in  muscle  fibers,  visible  to  the  naked  eye  as  fine, 
white,  opaque  filaments.  They  have  been  found  in  deer,  cattle,  sheep, 
swine,  rabbits  and  man,  and  occasionally  in  birds  and  reptiles.  Al- 
though long  known,  our  knowledge  of  them  is  still  defective.  In  sheep 
they  are  the  cause  of  severe  epizootics,  and  in  mice  they  are  fatal: 
otherwise  they  seem  to  be  harmless  parasites  and  in  rare  instances 
have  been  accidentally  found  at  autopsy  in  man. 

The  method  of  transmission  is  not  definitely  known.  Theobald 
Smith  was  able  to  infect  mice  with  Sarcocystis  m-uris  by  feeding  mus- 
cles from  infected  mice,  and  Darling  was  able  to  infect  guinea-pigs 
in  the  same  manner.  In  nature  it  is  probable  that  infection  occurs 
through  the  intestinal  tract.  They  are  exceedingly  common  in  some 
localities  and  may  be  seen  in  most  abattoirs;  they  luive  been  found  in 
ninety-eight  per  cent  of  swine,  ninety-eight  per  cent  of  sheep,  and 
commonly  in  deer  and  mice. 


1136  PATHOGENIC  PROTOZOA 

To  the  naked  eye  they  appear  as  whitish,  opaque,  cylindrical  bodies 
in  the  muscles  lying  parallel  to  the  fibres.  In  the  sheep,  Sarcocystis 
tenella  reaches  a  length  of  sixteen  mm.,  and  in  the  deer  cysts  of  fifty 
mm.  are  found.  In  structure  the  * '  tube ' '  is  seen  under  the  microscope 
to  be  composed  of  many  sickle-shaped  spores,  called  Rainy 's  cap- 
sules. The  tube  has  not  a  single  cavity  but  a  honey-comb  or  alveolar 
structure,  and  the  spores  are  found  in  small  aggregations  in  the  cham- 
bers, completely  walled  off  from  one  another.  The  tube  itself  has  a 
heavy  striated  wall,  either  secreted  by  the  organism  or  composed  of 
altered  muscle  fiber  of  the  host.  The  spores  are  sickle-  or  kidney- 
shaped  and  vary  both  among  themselves  and  with  the  host.  They 
contain  a  nucleated  trophozoit,  and  at  one  pole  either  a  clear  or  an 
obliquely  striated  body. 

Laveran  and  Mesnil6  (1899)  obtained  a  toxin  from  Sarcocystis 
tenella  and  named  it  sarcocystin.  Darling,  working  in  Panama,  found 
this  organism  in  the  biceps  muscle  of  a  negro  from  Barbados.  Not 
more  than  five  human  cases  are  known. 

8  Trop.  Diseases  Bull.  London,  1920,  16,  96. 


CHAPTER  LVII 

CLASS  IV— INFUSOEIA 
SUB-CLASS—  (CILIATA) 

THIS  class  is  sharply  distinguished  from  all  other  protozoa  by  the 
presence  of  numerous  cilia,  distributed  in  various  ways  over  the 
ectoplasm,  which  serve  as  organs  of  locomotion,  and  by  the  presence 
of  two  or  more  nuclei.  They  fall  into  two  classes,  the  ciliata,  which 
are  provided  with  cilia  during  the  entire  life  cycle,  and  the  suctoria, 
which  lose  their  cilia  on  entering  the  adult  stage; -the  latter  are  of 
no  interest,  since  none  are  holozoic. 

Only  one  of  the  ciliates  is  of  importance  in  medicine,  Balantidium 
coli.  Balantidium  minutum  and  Nyctotherus  fa~ba,  having  been 
reported  only  once  in  man  by  Schaudinn,  are  too  rare  to  be  con- 
sidered here. 


BALANTIDIUM  COLI  (Malstan) 

This  parasite,  first  described  in  1857,  is  commonly  found  in  the 
lower  intestinal  tract  of  swine ;  while  usually  harmless,  it  may  cause 
a  moderate  mortality  in  them  from  subacute  and  chronic  dysentery. 
It  has  frequently  been  reported  as  present  in  man,  and  is  readily 
diagnosed  on  examination  of  the  stools. 

The  organism  is  much  like  paramecium  and  is  actively  motile  when 
obtained  from  fresh  stools,  or  from  scrapings  from  the  ulcerated 
cecum  at  autopsy.  The  body  is  ovoid  and  rather  stumpy  toward  the 
anterior  end,  distinguished  by  the  triangular  peristome;  average 
measurements  are  about  eighty  by  sixty  microns.  The  ectoplasm  is 
covered  with  thick,  parallel  bands  of  active  cilia,  giving  the  animal 
a  striated  appearance;  the  macronucleus  is  kidney  shaped  and  lying 
close  to  it  the  micronucleus  may  usually  be  distinguished.  At  one 
side  of  the  organism  are  found  two  contractile  vacuoles,  and,  in  the 
endoplasm,  food  vacuoles  and  fat  droplets. 

1137 


1138 


PATHOGENIC   PROTOZOA 


Multiplication  in  its  simplest  form  consists  of  binary  division, 
though  conjugation  also  occurs.  In  the  feces  of  the  host  encysted 
forms  are  common,  and  by  means  of  these  the  infection  is  transferred 
to  new  hosts. 

Although  long  believed  to  be  harmless,  pathological  lesions  have 
been  found  by  Strong  and  others  in  man  and  monkeys,  consisting  of 


FIG.  164. —  BALANTIDIUM  COLI  IN  A  FOLLICLE  OF  THE  COLON,  BREAKING  THROUGH 
THE  MUCOSA.     (Army  Med.  School  Collection,  Washington,  D.  C.) 

thickening  and  ulceration  of  the  infected  intestine,  with  penetration 
of  the  balantidium  through  the  gut  wall  to  the  subserous  layer.  Exten- 
sive lesions  may  be  found  at  autopsy,  even  in  the  absence  of  a  his- 
tory of  dysentery  or  diarrhea. 

The  disease  is  transmitted,  in  all  probability,  by  ingesting  the 
cysts  from  swine  which  undergo  development  in  the  human  being  to 
the  cyst  stage:  this  stage,  however,  is  never  found  in  human  infec- 
tions and  man,  therefore,  is  probably  incapable  of  spreading  the 
disease. 


INFUSORIA  1139 


Treatment  has  not  been  satisfactory :  Brag,1  however,  has  reported 
a  case  which  responded  to  arsphenamme  after  simaruba,  iodoform 
enemas  and  oil  of  chenopodium  had  failed. 


CHLAMIDOZOA 

There  is  a  small  group  of  very  minute  intracellular  organisms 
which  are  believed  by  many  to  be  protozoa,  and  for  them  von  Pro- 
wazek  has  created  the  class  named  '  *  chlamydozoa. "  He  believes  that 
such  organisms  produce  smallpox,  vaccinia,  hydrophobia,  trachoma, 
scarlatina,  Molluscum  contagiosum,  avian  plague,  the  contagious  epi- 
thelioma  of  birds,  hoof  and  mouth  disease,  jaundice  of  silk  worms 
and  others.  The  delicate  organisms  are  first  noticed  as  fine  dots  of 
chromatin  lying  in  the  cytoplasm  of  the  infected  cell  (the  so-called 
elementary  bodies).  When  larger  they  are  called  initial  bodies  and 
are  believed  to  set  up  a  reaction  in  the  cell,  which  causes  an  extru- 
sion of  the  plastin  from  the  nucleus;  this  envelops  the  initial  bodies 
like  a  mantle,  hence  the  name  "  Mamydozoa,"  from  the  Greek  stem 
"chlamys,"  meaning  mantle.  Inside  of  this  covering  they  multiply, 
in  some  instances  invading  the  nucleus. 

Their  very  existence  is  a  matter  of  dispute,  yet  at  the  present  time 
the  consensus  of  belief  is  that  they  constitute  a  group  of  parasites 
and  are  not  merely  forms  of  cell  inclusions  or  degenerations. 

Noguchi2  has  shown  that  in  trachoma  these  organisms  may  be 
found  alone  without  any  other  pathogenic  organisms  being  present, 
and  that  they  may  be  transferred  to  the  conjunctiva  of  the  baboon 
and  higher  apes,  producing  a  mild  form  of  the  disease.  Smears  of 
the  exudate,  taken  from  the  infected  animals  and  stained  with  Giemsa, 
show  cell  inclusions  without  the  presence  of  bacteria. 

1Brug,  S.  L.,  Tropical  Diseases  Bulletin,  London,  1921,  17,  181. 
-Noguchi,  Jour.  Exp.  Med.,  N.  Y.,  1915,  xxii,  304. 


CHAPTER  LVIII 

TECHNIQUE  OF  BLOOD  EXAMINATIONS  FOE  PROTOZOA 

SLIDES  and  cover  glasses  must  be  scrupulously  clean,  and  are  best 
kept  in  covered  glass  jars  which  are.  dust-proof.  For  most  purposes 
stained  preparations  on  slides  are  satisfactory  and  much  easier  to 
handle  than  cover  glasses. 

In  all  investigations,  both  wet  and  dry  preparations  should  be 
made ;  some  practice  is,  of  course,  necessary  to  obtain  satisfactory  ones, 
thin  enough  to  show  details.  As  Manson  says,  in  looking  for  dates 
on  coins,  one  would  not  pile  one  coin  on  top  of  another ;  so,  in  search- 
ing blood  cells  for  parasites,  it  is  necessary  to  have  the  cells  lying 
flat,  and  in  a  single  layer.  Wet  preparations  are  made  with  a  drop  of 
blood  not  much  larger  than  the  head  of  a  pin,  on  which  a  cover  glass 
is  dropped,  and  gently  pressed  down;  it  is  advantageous  to  lute  the 
margin  of  the  cover  glass  with  warm  vaseline  (warmed  by  holding 
the  camel's  hair  pencil  over  a  flame  for  a  few  seconds)  to  prevent 
evaporation  and  consequent  currents  under  the  cover  glass.  A  prop- 
erly sealed  wet  preparation  may  be  examined  at  intervals  during 
several  days,  and  this  is  often  necessary  in  studying  movements  and 
the  life  cycle.  Malarial  parasites,  trypanosomes  and  others  are  quickly 
detected  by  their  movements. 

Stained  preparations  are  necessary  for  the  study  of  details  of 
structure,  and  for  clinical  work  are  invaluable,  since  smears  may  be 
collected  at  the  bedside,  and  examined  later  at  home,  either  by  day- 
light or  artificial  light.  In  general,  a  one  hundred  watt  concentrated- 
filament  nitrogen  bulb  will  be  found  the  best  source  of  illumination 
for  the  study  of  both  living  and  stained  protozoa.  The  Welsbach 
mantle,  acetylene  gas  or  even  a  kerosene  lamp  are  satisfactory  sub- 
stitutes. At  times  a  color  screen,  made  by  interposing  a  round,  glass 
flask,  filled  with  diluto  copper  sulphate  solution,  between  the  light  and 
the  mirror,  will  prove  advantageous.  It  is  particularly  in  the  South 
and  in  the  tropics  that  artificial  illumination  has  been  most  valuable. 
To  prepare  stained  preparations,  a  small  drop  of  blood  from  the 
^ar  is  placed  on  a  slide  near  one  end,  while  with  another  clean  slide 

1140 


BLOOD  EXAMINATIONS  FOR  PROTOZOA       .  1141 

the  spread  is  made  by  holding  its  narrow  end  in  the  drop  until  the 
blood  has  flowed  between  the  two,  and  then  pushing  the  spreader,  or 
second  slide,  held  at  an  angle  of  about  45°,  toward  the  opposite  end 
of  the  first  slide.  The  drop  of  blood  follows  closely  behind  the 
spreader,  and  the  thickness  of  the  film  may  be  varied  by  changing  the 
angle  of  the  spreader.  The  thin  film  of  blood  dries  quickly  in  the 
air.  It  is  never  fixed  in  the  flame,  but  always  in  methyl  alcohol  or  in 
the  stain  itself. 

Many  varieties  of  blood  stains  have  been  proposed,  but  the  simplest 
and  best  are  those  of  Wright  and  MacNeal.  Wright's  stain  is  pre- 
pared as  follows: 

Dissolve  0.5  gm.  of  sodium  bicarbonate  in  100  c.c.  distilled  water, 
and  add  1  gm.  of  methylene  blue  (Gruebler).  Any  of  the  methylene 
blues  of  Gruebler  known  as  "BX,"  Koch's  or  Ehrlich's  rectified  may 
be  used.  It  seems  to  be  important  that  the  bicarbonate  of  soda  be  all 
dissolved  before  adding  the  methylene  blue. 

The  mixture  is  next  to  be  steamed  in  an  ordinary  steam  sterilizer 
for  one  hour,  counting  the  time  after  "the  steam  is  up."  The  heating 
should  not  be  done  in  a  pressure  sterilizer,  or  in  a  water-bath,  or  in 
any  other  way  than  as  stated.  This  steaming  of  the  alkaline  solution, 
of  methylene  blue  effects  certain  changes  in  the  methylene  blue  where- 
by a  polychromatic  quality  is  given  to  it,  so  that  the  compound  with 
eosin,  which  is  later  to  be  formed  with  it,  has  the  property  not  only 
of  differentially  staining  the  chromatin  of  the  malarial  parasite,  but 
also  of  differentiating  and  bringing  out  more  sharply  the  nuclei  and 
granules  of  the  white  blood  corpuscles. 

When  the  steaming  is  completed,  the  mixture  is  removed  from  the 
sterilizer  and  allowed  to  cool,  the  flask  being  placed  in  cold  water  if 
desired.  When  it  is  cold,  without  filtering,  pour  it  into  a  large  dish 
or  flask,  and.  add  to  it,  stirring  or  shaking  meanwhile,  a  sufficient 
quantity  of  a  1:1000  solution  of  eosin  (Gruebler,  yellowish,  soluble 
in  water)  until  the  mixture,  losing  its  blue  color,  becomes  purplish, 
and  a  scum  with  yellowish  metallic  luster  forms  on  the  surface,  while 
on  close  inspection  a  finely  granular  black  precipitate  appears  in 
suspension.  This  will  require  about  500  c.c.  of  the  eosin  solution  for 
100  c.c.  of  alkaline  methylene  blue  solution.  (The  proper  amount 
of  eosin  to  add  may  be  determined  by  placing  a  drop  of  mixture  on 
white  filter  paper,  and  adding  eosin  until  the  bine  spot  shows  a  dis- 
tinct halo  of  pink.) 

The  precipitate  is  collected  on  a  filter,  and,  without  washing,  is 


1142  PATHOGENIC   PROTOZOA 

allowed  to  dry  thereon ;  when  thoroughly  dry,  dissolve  this  precipitate 
in  pure  methyl  alcohol  in  the  proportion  of  three-tenths  of  a  gram  to 
one  hundred  cubic  centimeters  of  alcohol.  This  alcoholic  solution  is 
the  staining  fluid. 

MacNeal1  has  shown  that  both  methylene  azure  and  methylene 
violet  participate  in  the  nuclear  staining,  and  that  an  excellent  stain, 
equal  to  any,  may  be  made  directly  by  mixing  the  pure  dyes  accord- 
ing to  the  following  formula: 

Solution  A.     Methylene  azure    0.3  gm. 

Methylene  violet   (Bernthsen's,  insoluble  in  water) 0.1 

Methylene  blue    2.4 

Itt ethyl  alcohol,  pure   (Merck's  reagent) 500.0 

Solution  B.     Eosin,  yellowish,  water  soluble 2.5 

Methyl   alcohol,   pure 500.0 

These  stock  solutions  will  keep  for  at  least  a  year.  They  are  mixed 
as  needed  in  equal  parts,  and  diluted  by  the  addition  of  25  c.c.  of 
methyl  alcohol  to  each  100  c.c.  of  the  mixture.  The  mixture  will 
keep  for  several  months. 

The  method  of  staining  is  the  same,  whatever  methyl  alcohol  stain 
be  used.  The  cover  glass  or  slide  is  flooded  with  the  stain,  which  is 
allowed  to  act  for  one  minute ;  next,  as  much  distilled  water  is  added, 
drop  by  drop,  as  the  slide  will  hold,  or  until  a  yellowish  metallic 
scum,  is  formed  on  the  surface,  and  the  mixture  is  allowed  to  act 
from  three  to  five  minutes.  A  convenient  staining  dish  is  made  by 
laying  a  pair  of  glass  rods  on  top  of  a  flat  oblong  dish ;  the  rods  may 
be  held  in  place  with  adhesive  plaster  or  perforated  strips  of  wood. 
Fixation  is  accomplished  by  the  undiluted  solution,  but  the  actual 
staining  does  not  occur  until  water  is  added ;  by  repeated  washing  in 
distilled  water,  any  desired  differentiation  may  be  made. 

It  is  sometimes  convenient,  when  searching  for  malarial  parasites 
in  clinical  cases,  to  stain  as  follows  :2 

Use  four  bottles  or  Coplin  jars;  in  the  first,  put  the  undiluted 
stain,  and  in  it  immerse  the  slide  for  one  minute ;  in  the  second,  put 
distilled  water  and  transfer  the  slide  to  it  for  four  or  five  minutes; 
in  the  third  jar  put  diluted  Manson's  stain,  about  0.5  c.c.  to  50  c.c. 
water,  and  in  this  the  smear  remains  one-half  minute;  in  the  fourth 


1  MacNeal,  Jour.  Inf.  Dis.,  Chicago,  1906,  iii,  412. 

*  Russell,  F.  F.,  Jour.  Amer.  Med.  Ass.,  Chicago,  1915,  Ixiv,  2131. 


BLOOD  EXAMINATIONS   FOR   PROTOZOA  1143 

bottle  or  tumbler  put  distilled  water,  in  which  the  slide  is  washed 
quickly  and  then  dried.  This  method,  for  the  average  man,  is  more 
economical  and  simpler  than  tjie  open  method.  Stained  smears  are 
examined  directly  in  immersion  oil  without  the  use  of  cover  glass. 

Hanson's  stain  is  prepared  as  follows: 

Two  grams  of  methylene  blue,  medicinally  pure  (Ilochst),  is 
added  to  100  c.c.  of  a  boiling  5  per  cent  solution  of  borax.  This  stain, 
though  not  permanent,  will  last  a  long  time.  It  is  used  chiefly  for 
the  diagnosis  of  malaria.  It  is  diluted  before  use.  Stain  for  ten  to 
fifteen  seconds. 

When  parasites  are  few,  as  in  latent  malaria,  thick  films  may  be 
used,  as  first  proposed  by  Ross.  A  large  drop  of  blood  is  spread 
thickly  on  the  side,  with  the  needle  or  pen  used  in  puncturing  the 
ear;  after  drying,  it  is  put  into  (1)  95  per  cent  methyl  alcohol  to 
which  1  per  cent  of  hydrochloric  acid  has  been  added,  until  the  smear 
has  been  hemolized;  (2)  it  is  then  thoroughly  washed  in  running 
water,  (3)  and  then  stained  in  the  usual  way  with  a  methyl  alcohol 
stain.  Although  the  method  takes  considerable  practice,  it  is  a  valu- 
able procedure. 


INDEX  OF  AUTHORS 


ABBOTT,  116,  389 

ABEL,  240,  724,  726,  810 

ACHARD  AND  BENSAUDE,  688 

ADAMI,  222,  631 

ADAMI  AND  CHAPIN,  1022 

ADIE,  HELEN,  1099 

ALBRECHT  AND  GHON,  518,  523,  566,  808 

ALVAREZ  AND  TAVEL,  618 

AMAKO,  712 

ANDERSON,  596,  1035 

ANDERSON  AND  FROST,  916,  918 

ANDERSON   AND  GOLDBERGER,   923,  937, 

939 

ANDERSON  AND  MCCLINTIC,  99 
ANDREWES  AND  HARDER,  409 
ANTHONY,  419 
ARISTOTLE,  2 
ARKWRIGHT,  BACOT  AND  DUNCAN,  943, 

949 

ARLOING,  77 

ARLOING,  CARNEVIN  AND  THOMAS,  771 
ARMSTRONG,  746 
ARNHEIM,  941 
ARNING,  624 

ARONSON,  108,  166,  411,  425,  430,  451 
D'ARSONVILLE  AND  CHARRIN,  79 
ARTHUS,  355 
ARUSTAMOFF,  963 

ASCOLI   AND  FlGARI,   253 
ASHBURN    AND    CRAIG,    931 
ASHFORD,   991 

ASOKAWA,  268 

AVERY,  156,  447,  497 

AVERY,  CHICKERING,  COLE  AND  DOCHEZ, 

439,  458,  462 
AVERY  AND  DOCITEZ,  455,  463 

AXENFELD,  50S 


BABES,  11,  563,  625,  868,  869,  902,  967 
BABES  AND  LEPP,  248 


BACOT,  957 

BAGINSKY,  568,  734 

BAGINSKY  AND  SOMMERFELD,  412,  419, 

425,  926 
BAIL,  349,  394 
BANDELIER  AND  KOEPKE,  611 
BANDI  AND  SIMONELLI,  852 
BANG,  693,  799,  1035 
BALDWIN,  372 
BARBER,  178,  750 
BARKER  AND  COLE,  667 
BARSIEKOW,  164 
BASCHETTI,  1035 
BASENAU,  688,  1033 
BASS,  C.  C.,  1116 
BASSETT-SMITH,  540,  437,  795 
BAUMGARTEN,  125,  593 
BECHMANN,  95 
BECK,  563,  566 

BEHRING  AND  KITASATO,  248,  250 
BEIIRING,  93,  95,  98,  246,  257,  274,  353, 

580,  778 

BEHRING  AND  WERNICKE,  248,  250 
BEIJERINCK,  28,  30,  1031,  1044 
BELFANTI,  729 
BELFANTI  AND  CARBONE,  252 
BELPAEFF,  249 
BENDA,  96 
BENGSTAN,  945,  755 
BENGER,  521 
BENIANS,  120 
BERANECK,  606 
BERESTNEFF,  964 
BERESTNEW,  961,  970 
BERTARELLI,  858 
T.ERTHELOT,  61 

BERTRAND  AND  WEISWETLLER,  1046 
BESSATT,  709 

BESREDKA,  369,  657,  684 
DEBEURMANN  AND  GOUGEROT,  975,  993 


1145 


1146 


INDEX  OF  AUTHORS 


BEZANCON,  399 

BEZANCON,  GRIFFON  AND  LESOURD,  511 

BIENSTOCK,  228,  587,  632 

BIGGS  AND  PARK,  284 

BILLROTH,  7 

BlRCH-HlRSCHFELD,   632 

BITTER,  35,  201,  618 

BLAKE,  965 

BLAKE  AND  CECIL,  459 

BLAISE  AND  SAMBAC,  78 

BLAKE  AND  TRASK,  923 

BLANC  AND  POZERSKI,  761 

BLISS,  928 

BLOCK,  B.,  AND  MASSINI,  999 

BLUMENTHAL,  672 

BLUMER,  655 

BOGART  AND  BERNARD,  252 

BOGGS,  499 

BOLLINGER,  966 

BOLTON,  26 

BONNEY  AND  BROWNING,  97 

BORDET,   252,   264,   278,   279,   290,   293, 

294,  507 

BORDET  AND  CUICA,  72,  74 
BORDET  AND  GENGOU,  298,  315,  505 
BORDONI-UFFREDUZZI,  449 
BORISSOW,  393 

VON   DEM   BOME,   870 

BOSTROEM,  968 

BOYCE  AND  WOODHEAD,  1042 

BOYLE,  EGBERT,  3 

BRADFORD,  BASHFORD  AND  WILSON,  487 

BRANELL,  6 

BRAU  AND  DENIER,  837 

BRAUN,  425,  427 

BREM,  596 

BRIEGER,  234,  656 

BRIEGER  AND  BOER,  573,  732 

BRIEGER  AND  COHN,  731,  732 

BRIEGER  AND  FRANKEL,  573 

BRIEGER  AND  KEMPNER,  743 

BRILL  AND  LIBMAN,  805 

BROADHURST,  409,  435 

BROWNING,  GULBSAUEN,KENNAWAY  AND 

THORNTON,  96 
BROWNLEE,  492 
BRUCE,  796,  1090 
BRUDZINSKI,  1045 


BRUG,  1139 

BRUMPT,  943,  984,  1091 

v.  BRUNN,  107 

BUCHNER,   22,    77,    250,    254,    277,   278, 

628,  1018 

BUCHNER  AND  HAHN,  606 
BUCHNER  AND  MEISENHEIMEK,  57 
BUDD,  643 
BUDINGER,  391 

BUERGER,    117 
BUFFON,  4 

BULL,  286 

BULL  AND  PRITCHETT,  648,  754 

BULLOCK  AND  ATKIN,  337,  339 

BULLOCH  AND  HUNTER,  806 

BULLOCK  AND  WESTERN,  337 

BULLY,  676 

BUMM,  547,  548 

BURGER  AND  WYNTENBERG,  434 

BURGERS,  120 

BURKE,  743,  745 

BURKHOLTZ,  77 

BURNET  AND  WEISSENBACH,  163 

BUSSE,  984 

BUTSCHII,  11,  12 

BUXTON,  686,  1063 

BUXTON  AND  COLEMAN,  214,  689 

BYAM,  949 

CALAMIDA  AND  BERTARELLI,  222 

CALKINS,  892,  904 

CALMETTE,  250,  255,  607 

CALMETTE  AND  GUERIN,  895 

CANFORA,  223,  735 

CANTANI,  498 

CANTACUZENE,  335 

CAPPS  AND  MILLER,  421,  1034 

CARLO  AND  EATTONE,  727 

CARREL,  DAKIN,  DAUFRESNE,  DEHELLY, 

AND  DUMAS,  93 
CARRIERS,  54 
CARTER,  971 

CASTELLANI,  288,  651,  870,  871 
CASTELLANI   AND  CHALMERS,  712,   795, 

813,  814,  842,  931,  984 
CAULFIELD,  754 
CECIL  AND  AUSTIN,  481 
CELLI,  78 


INDEX  OF  AUTHORS 


1147 


CERTES,  39,  848 

GHAMBERLAND  AND  Koux,  246,  779,  780 

CHARRIN,  79,  802 

CHARRIN  AND  KOGER,  282 

CHANTEMESSE,  668,  684 

CHANTEMESSE  AND  WIDAL,  655,  659 

CHAUVEAU,  246,  784 

CHESNEY,  456 

CHIARI,  671 

CHICK,  89 

CHECKERING,  466 

CHRISTEN,  82,  83 

CHUDIOKOW,  29 

CHURCHMAN,  170 

CHRISTMAS,  553 

CITRON,  327,  351 

CLAIRMONT,  724 

CLARK,  BAYARD,  559 

CLARK,  W.  M.,  146 

CLARK  AND  FENTON,  425 

CLARK  AND  LUBS,  138,  140,  141 

CLARKE,  892 

CLASS,  926 

CLEGG,  621 

CLOUGH,  467 

COBRETT,  583 

COCA,  353,  363,  371 

COGNIARD-LATOUR,  3,  6 

COHENDY,  1046 

COHN,  5,  41,  43 

COLE,  370,  445,  455 

COLE  AND  LLOYD,  149 

COLEBROOK,  971 

COLEMAN  AND  BUXTON,  651 

COLEMAN  AND  SHAFFER,  227 

COLES,  619 

CONN,  679,  1030,  1031,  1042,  1043 

CONRADI,  163,  651,  675,  708,  714,  1032 

CONRADI  AND  DRIGALSKI,  158 

COOKE  AND  VANDERVEER,  370 

CORRIL  AND  BABES,  880 

COUNCILMAN,  MAGRATH  AND  BRINCKER- 

HOFF,  892 
COUNCILMAN,    MALLORY    AND    WRIGHT, 

515,  516 

COURMONT  AND  GOYEN,  753 
CRAIG,  1131 
CRAIG  AND  NICHOLS,  860 


CRANES,  22 

CREEL,  817 

CREEL,  FAGOT  AND  WRIGHTSON,  109,  110 

CREITE,  735 

CRUM,  923 

CRUZ,  OSWALDO,  883 

CUICA,  72,  74 

GUSHING,  689 

GUSHING  AND  LIVINGOOD,  631 

CURTIS,  984 

DAKIN,  90,  92,  93 

DALE,  361 

DANSYZ,  265 

DARLING,  992 

DAVAINE,  6,  773 

DAVIES  AND  WELDON,  948 

DAVIS,  422,  435 

DAVIS  AND  EOSENOW,  421 

DEAN,  338,  625 

DEBRAND,  731 

DELEZENNE,  252 

DENEKE,  845 

DENYS,  430,  604,  606,  611 

DENYS  AND  LACLEF,  336 

DENYS  AND  MARCHAND,  430 

DENYS  AND  VAN  DE  VELDE,  395 

DESLONGCHAMPS,  389 

DEUTSCH  AND  FEISTMANTEL,  349 

DICKSON,  742,  745,  746,  747 

DlCKSON   AND  HOWITT,   747 

DIEUDONNE,   77,  78,   166,   775,   269,  833 

DOBELL,  CLIFFORD,  1056 

DOCHEZ,  AVERY  AND  L.ANCEFIELD,  412, 

419,  432 

DODDS-PRICE,  1096 
DOERR,  709,  352,  649 
DOERR  AND  EUSS,  360 
DOCHEZ,  467 
DOCHEZ  AND  AVERY,  474 
DOCHEZ  AND  GILLESPIE,  446 
DONATH  AND  L.ANDSTEINER,  300 
DONITZ,  272,  239 
DOPTER,  522 
DORSET,  24 
DORYLAND,  27 
DRAPER  AND  HANFORD,  927 
DREYER  AND  MADSEN,  261 


1148 


INDEX  OF  AUTHORS 


DRIGALSKI,  670 

DRIGALSKI  AND  CONRADI,  652,  670 

DORR,  674 

DRURY,  601 

DUBARRE  AND  TERRE,  615 

DUCLAUX,  1043 

DUJARRIC   DE   LA    RlVIERE,   486 

DUNBAR,  370 

VON  DUNGERN,  252 

DUNHAM,  523,  560,  686,  694,  833 
DUTTON  AND  TODD,  849,  863,  865 
DURHAM  AND  MYERS,  880 
DUSCH,  4 
DUVAL,  621,  622 
DUVAL  AND  SHARER,  715 

EBERTH,  643 

ECONOMO,  919 

EHRENBERG,  2 

EHRLICH,  124,  238,  250,  255,  256,  257, 

258,  262,  263,  266,  587,  734 
EHRLICH,  KOSSEL  AND  WASSERMANN,  258 
EHRLICH  AND  MORGENROTH,  279,  299 
EISENBERG,  44,  806 

ElSENBERG  AND   VOLK,   662 

V.  ElSLER  AND  PRIBRAM,  729 

ELSER,  527 

ELSER  AND  HUNTOON,  519,  523,  533 

ENDO,  159 

ENGELMAN,  28,  68 

EPPINGER,  782,  964 

EPSTEIN,  94,  213,  651 

VAN  ERMENGEN,  14,  119,  740,  688 

ERNST,  804,  11,  1035 

v.  ESMARCH,  77,  82,  177,  180 

ESCHERICH,  224,  628,  228,  564,  1045 

EVANS,  799 

EVANS  AND  RUSSELL,  109 

EWING,  852,  893 

EXNER  AND  HEYWARSKI,  674 

EYKMANN,  54,  55 

EYRE,  198,  419,  795,  797,  1033 

FABER,  424 
FAIRLEY,  945 
FARNET,  656 
FAURE-BEAULIEU,  552 


FEHLEISEN,  401,  414 

FEJES,  487 

FERMI  AND  PERNASSI,  732 

FERRAN,  31,  246,  729,  841 

FERRI  AND  CELLI,  78 

FERRY,  798 

FICKER,  76,  283,  666 

FIEDLER,  885 

FIELD,  927 

FINGER,  789,  794 

FINGER  AND  LANDSTEINER,  859 

FlNLAY,   875 

FINK,  357 

FlNKELSTEIN,    805 
FlNKLER  AND  PRIOR,  845 

FIRTH  AND  HORROCK,  1014 

FISCHER,  A.,  9,  10,  19,  24,  26,  64,  66, 

388 

FISCHER  AND  PROSKAUER,  106 
FLANDRIN,  693 

FLEISCHNER,  MEYER  AND  SHAW,  800 
FLEMING,  33 
FLEXNER,  522,  526,  531,  533,  656,  701, 

709,  964 

FLEXNER  AND  AMOSS,  917 
FLEXNER,  CLARKE  AND  DOCHEZ,  917 
FLEXNER  AND  JOBLING,  529,  530 
FLEXNER  AND  LEWIS,  914 
FLEXNER  AND  NOGUCHI,  423,  732,  915 
FLUGGE,  103,  104,  388 
FOA,  456 

FOA  AND  CARBONE,  454 
v.  FODAR,  249,  277 
FOOT,  951 
FOOTE,  679 
FORD,  689 
FOGES,  734 
FORNAECA,  399 
FORNET  AND  MULLER,  305 
FORSSMAN  AND  LUNSTROM,  747 
FORSTER,  F.,  36 
FOSTER,  437 

FOSTES  AND  COOKSON,  491 
FRAENKEL,  619,  752,  1013,  1014 
FRANKEL,  A.,  440,  456,  464,  655,  664, 

720,  779,  649 
FRANZOTT,  389 
FRASER,  596 


INDEX  OF  AUTHORS 


1149 


FREER,  78 

FREUNDENREICH,  1042,  1043 

FINEMAN,  990 

FRIEDBERGER  AND  HARTOCH,  361,  365 

FRIEDLANDER,  439,  720 

FRIEDMANN,  616,  226,  362 

FRIEDRICH,  490 

FRISCH,  724 

FROCASTOR,  2 

FROSCH  AND  KOLLE,  405 

FROST,  489 

FUHRMANN,  49 

FULLER,  1019 

GABBET,  125,  588 

GAETHGENS,  674 

GAFFKY,  80,  643 

GAFFSKY,  398 

GALTIER,  794 

GAMALEIA,  844 

GARBAT,  673 

GARBAT  AND  MEYER,  685 

GARRE,  391 

GARTNER,  676,  687 

GATES,  518 

GAUTHIER,  1076 

GAVINA  AND  GERARD,  939,  943 

GAY,  296,  379,  419,  668 

GAY  AND  CHICKERING,  468 

GAY  AND  CLAYPOLE,  650,  685 

GAY  AND  SOUTHARD,  359 

GEPPART,  105 

GENGOU,  294,  296,  327 

VAN  GEHUCHTEN,  902 

GERONNE,  676 

GESSARD,  802,  804 

GHEARGHIEWSKI,  806 

GHON  AND  H.  PFEIFFER,  559 

GHON  AND  SACHS,  756 

GIBSON,  272 

GIBSON  AND  COLLINS,  272 

GlEBERT,    688 
GlEMSA,    127 

VAN  GIESON,  903 

GlLCHRIST,  984 

GLOBIG,  37 
GLOVER,  541 
GMINDER,  693 


GOLDBERGER,     WILLIAMS    AND    HACKTEL, 

575 

GORDON,  153,  522,  540,  409 
GORDON,  FLACK  AND  HINES,  520 
GORGAS,  875,  883 
GOTTSCHLICH,  25,  942 
GOTTSTEIN,  79 
GOULD,  756 
GRAHAM,  931,  946 
GRAM,  56,  120 
GRAMENITSKI,  295 
GRASSBERGER,  498 
GRATZER,  946 

GRAUCHER  AND  LEDOUX-LEBARD,  593 
GREKOFF,  1046 
GRIFFITH,  971 
GROSS,  848 
GRUBER,  81,  95,  664 
GRUBER  AND  DURHAM,  251,  282,  288 
GRUNHAGEN,  892 
GUARNIERI,  445,  449,  892 
GUERIN,  897 
GUITERAS,  877 
GUMPRECHT,  733 

GUNTHER,  439 

GWYN,  688 

HAAS,  80 

HAFFKINE,  841 

HAHN,  242,  243,  574,  657 

HALL,  919 

HALLIER,  7 

HAMBURGER,  986,  993,  1034 

HAMBURGER  AND  SLUKA,  595 

HAMMERSCHLAG,  603 

HAMMERSTEIN,  53 

HANEL,  389 

HANKIN  AND  LEUMANN,  21 

HANKIN  AND  WESTBROOK,  202 

HANSEN,  982,  619 

HARDING  AND  OSTENBERG,  159 

HARRIS,  909 

HARRIS  AND  SHACKELL,  908 

HARRISON  AND  GALTIER,  1044 

TlAUSEB,  640 

HEIM,  1032 
HEIMAN,  548,  552 
HEINEMANN,  1030,  1034 


1150 


INDEX  OF  AUTHORS 


HKKTOKX,  UL'L',  338,  428 

llEKTOEN   AND  PERKINS,  993 

HEKTOEN  AND  KUEDIGER,  337 

HELLER,  758 

HELLRIEGEL  AND  WILFARTH,  62,  63 

VAN  HELMONT,  4 

HENLE,  3 

HENRY,  753 

HENRY  AND  LACY,  763 

HENRIJEAN,  730 

D  'HERELLE,  70,  71 

HERMANIES,  551 

HERRICK,  538 

HERRINGHAM,  946 

HERTER,  224,  211,  634 

HESSE,  592,  1033 

HEYFELDER,  490 

VON  HIBLER,  750 

VON  HIBLER,  KOLLE  AND  WASSERMANN, 

770 
HILBERT,  35,  572 

HlLDERBRANDT,    221 

HILL,  105,  112,  1021 
HINE,  524 
HIRSCH,  807,  936 

HlRSCHBERGER,    1035 

Hiss,  13,  116,  346,  403,  405,  406,  433, 
444,  445,  449,  453,  467,  652,  654,  704, 
705,  946 

Hiss  AND  ATKINSON,  249,  272 

HlSS  AND  EUSSELL,  703 

HOFER,  726 

HOFFMANN,  287 

HOFFMANN-WELLENHOFF,  582 

HOGYES,  907,  909 

HOLLER,  347 

HOLMAN,  409 

HOLMES,  5 

HOLST,  688 

HOOKER,  397 

HOPKINS  AND  LANG,  409 

HORTON-SMITH,  652,  654 

HOUSTON,  409,  673,  1013,  1024 

HOUSTON  AND  THOMAS,  675 

HOWARD,  724 

HOWARD  AND  PERKINS,  448 

HOWE,  922 

HUBER  AND  KOESSLER,  363 


llUDDLESTON,   897 
HUDDLESTON,   800 

HUEPPE,  827 
HUEPPE  AND  WOOD,  785 
HUNTEMULLER,  1018 
HUNTOON,  33,  149 

ICHIKAWA,  685 

IDO,  HOKI,  ITO  AND  WONI,  887 

INADA,  885,  886 

INADO  AND  IDO,  888 

IROUS  AND  MARINE,  420 

IRWIN  AND  HOUSTON,  675 

ISRAEL,  966 

IWANOFF,  24 

IWANO,  778 

JACKSON,  163,  816 

JACKSON  AND  MELIA,  164 

JAEGER,  515 

JAEHLE,  499 

JAGER,  724 

JANOWSKI,  77 

JAVORONE,  1095 

JENNER,  127 

JENSEN,  122 

JOACHIM,  249 

JOAS,  287 

JOBLING  AND  PETERSEN,  247,  366 

JOHNSTON,  649 

JONES,  23,  88 

DE  JONG,  293 

JORDAN,  51,  690 

JORDAN  AND  HEINEMANN,  1028 

JORDAN  AND  IRONS,  1020 

JORDAN  AND  SHARP,  493 

JORGENSEN  AND  MADSEN,  287 

JUNGMANN,  949 

KABESHIMA,  72 

KALBERLAH,  676 

KAMEN,  633 

KANEKO  AND  OKUDA,  873 

KAPPES,  22 

KARLINSKY,  654 

KASTLE  AND  ELVOVE,  159 

KEMPNER,  250,  747 

KEMPNER  AND  POLLACK,  744,  745 


IXDKX    DP    .UTHOK.S 


1151 


KEMPNEB  AND  SCHEPILEWSKY,  268 
KENDALL,  160,  _.-.  __  • 
KENDALL  AND  DAY.  __~ 
KENDALL,  DAY  AND  WALKER,  642 
KINYOUN,  899 

KlNSELLA  AND  SWIFT,  411,  412 

KlRCHER,    1 

KlSTER  AND  WOLFF,  292 

KITA-  -_7.  729,  732,  770,  807,  810 

KlTASATO  AND  WEYL,  29 

Km,  7~_ 

KITT  AND  MAYS,  829 

KLEBS,  7,  439,  880,  561,  607 

KLEIN,  211,  677,  587,  649,  667 

KLEMPERER,  618,  454 

KLIGLER,  165 

KLINGLER,  971 

KLINGMULLER  AND  BAERMANN,  861 

KLOTZ,  1046 

.  7o4 

KNAPP,  583 
KNOEPFELMACHER,  914 
KNORR,  257,  353 
ROBERT  AND  STILLMARCK,  256 
KOCH,  3,  7,  8,  77,  86,  94,  104,  105,  106, 

401,  439,  502,  590,  604,  605,  606,  607, 

610,  611,  612.  649,  670,  756.  " 

836;  838,  865,  866,  1023 
Kocx,  GAFFKY  AND  LOEFFLER,  80,  81, 

783 

KOCH  AND  PETRUSCHKT,  429 
KOCH  AND  KABINOVITSCH,  615 
KOCH  AND  WOLFFHUGEL,  80,  779 
KOESSLLER  AND  HANKE,  52 
KOHER,  598,  600 
KOLLE  AND  HETSCH,  847 
KOLLE  AND  OTTO,  396 
KOLLE  AND  SCHUMANN,  841 
KOLLE  AND  WASSERMANN,  523,  616 
KOLMER,  894 

KOLMER    AND   STRICKLER,    999 
KORBRIDE,   455 

KORN,  617 
KORSCHUN,  346 
KOSSEL.  257 

CD  OVERBACK,  812 

T.-L,  WEBER  AND  HUESS,  1036 
KRAUNHALS,  805 


KRAUS,   251,   289,   292,   394,   667. 

685,  708,  837,  844 
KRAUS,  B.,  304 
KRAUS  AND  DOERR,  710,  708 
KRAUS  AND  Low,  286,  636 
KRAUS  AND  MEINICKE,  916 
KRAUS  AND  v.  PIRQUET,  291 
KRAUS  AND  STENITZER,  657 
KRAUSE,  372 
KRESLING,  7'Jl 
KRETZ,  270 

KRONIG  AND  PAUL,  87,  89,  96,  98 
DE  KRUIF,  751 
DE  KRUIF  AND  IRELAND, 
KRUMWIEDE,  870 

KRUMWIEDE  AND  KOHN,  689,  695 
KRUMWIEDE,    KOHN    AND    VALENTINE, 

648 

KRUMWIEDE  AND  PRATT,  170 
KRUMWIEDE,  PRATT  AND  KOHN,  690 
KRUSE,  349,  437,  701,  702,  710,  828 

KURTH,  689 

KUSONOKI,  999 

KUSTER,  i__    __      672 

KUTSCHER,  415,  670 

KUTSCHER,  KOLLE  AND  WASSERMANN, 

KUTSCHERT  AND  NOSSER,  584 
LAMAS  AND  MELTZER,  457 

L-\NDMANN,  743 

,-STEINER,   252 

LANDSTKINER  AND  JAGIC,  294 
LANDSTEINEB  AND  LEVADITI,  914 

JSTEINER   AND   POPPER,  913,  914 

LANDSTEINKR,   LEVADITI    AND    PRASEK, 

927 

LASSAR,  857 
LARREY,  885 
LAUBENHEIMER,  672 
LAVERAN,  847 

LAWS  AND  ANDERSON,  1022 
LEACH,  23 

VAN  LEEUWENHOEK,  2,  4 
LECHNER-SANDOVAL,  961 
LECLAIN  AND  VALLEE,  771 
LEDDERHASE,  804 


1152 


INDEX  OF  AUTHORS 


LEICHTENSTERN,  515,  489 

LEISHMAN,  336 

LENTZ,  107,  697,  703 

LENTZ  AND  HUNTEMULLEII,  916 

LEPIERRE,  526 

LESAGE,  1044 

LESCHKE,  487 

LEUCHS,  743,  747 

LEVADITI,  332,  338,  855 

LEVADITI  AND  BANKOWSKI,  854 

LEVADITI  AND  HARRIER,  901 

LEVADITI  AND  INMANN,  338 

LEVADITI  AND  MANOUELIAN,  871 

LEVADITI  AND  PETRESCO,  852 

LEVENE,  23 

LEVY,  925 

LEVY  AND  ALEXANDER,  420 

LEWITH,  80 

LIBMAN,  405,  689,  698 

LlBMAN  AND  ROSENTHAL,  453 

LlBORIUS,    180 
LlEBERMEISTER,    588 

V.  LINGELSHEIM,  393,  403,  430,  535,  407 

LISTER,  6,  480,  448 

LISTER  AND  TAYLOR,  487 

LISTON,  825 

LLOYD,  33 

LOEB,  386 

VAN  DER  LOEFF,  892 

LOEFFLER,   561,    571,    14,    80,    81,    119, 

129 

LOEFFLER  AND  FROSCH,  933 
LOEFFLER  AND  SCHUTZ,  787 
LOEWE  AND  STRAUSS,  921 
LOEW,  27 
LOHLEIN,  337 
LONGCOPE,  697 
LOWY,  940 

LO  WEN  STEIN,    615 
LUBARSCH,   241 
LUBBERT,   389 

LUDKE,  685 

LUERSEN   AND   KUHN,    1046 
LUSTGARTEN,   618,   850 

LUSTIG  AND  GALEOTTI,  825 
LYNCH,  KENNETH,  1074 

MAASEN,  59 


MACCALLUM,  416,  964 

MACCALLUM,  COLE  AND  DOCHEZ,  420 

MACKENSIE  AND  LONGCOPE,  368 

MATHERS,  423 

MACFAYDEN,  454 

MACFAYDEN  AND  ROWLAND,  657 

MACNEAL,  491,  988,  1142 

MADISON,  500 

MADSEN,  271 

MAFUCCI,  587,  604,  614 

MALLORY,  927,  968 

MALLORY  AND  HORNER,  507 

MALLORY  AND  MEDLAR,  927 

MALLORY  AND  WRIGHT,  132,  639 

MANSON-BAHR,  1097 

MANWARING,  363 

MARAGLIANO,  612 

MARBAIX,  414,  407,  416 

MARCHIAFAVA  AND  CELLI,  515 

MARCHOUX  AND  SALIMBENI,  871 

MARCHOUX,    SALIMBENI    AND    SIMONDP 

877,  884 

MARCHOUX  AND  SIMOND,  878,  879 
MARINESCO,  744 
MARKS,  916 
MARMIER,  425 
MARMOREK,  411,  425,  612 
MARSCHAL,  514 
MARTIN,  5,  72,  771 
MARTIN  AND  CHERRY,  256 
MARTINI  AND  LENTZ,  702 
MARX,  431,  909 
MASSOL,  1046 

MATHERS  AND  HERROLD,  540 
MAYER,  745 

MAYER  AND  AHREINER,  673 
MCCLINTIC,  99,  951 
McCoY,  625,  815,  816,  818 
McCoY  AND  CHAPIN,  826 
McGowAN,   798,   801 

MclNTOSH   AND   FlLDES,    187,    749 

McLEOD,  427 

McNEE,  BRUNT  AND  RENSHAW,  947 

MELENEY  AND  RAY,  541 

MEISENHEIMER,  57 

MENNES,  454,  467 

MESNIL,  286,  992 

MESSEA,  16 


INDEX  OF  AUTHORS 


1153 


MESSERSCHMIDT,  715 

METCHNIKOFF  's  BACTERIOTHERAPY,  1044 

METCHNIKOFF,  239,  250,  252,  278,  282, 

330,  762,  958,  1045 
METCHNIKOFF     AND     BESREDKA,     650, 

683 

METCHNIKOFF  AND  Eoux,  857 
METCHNIKOFF,   Eoux    AND   SALIMBENI, 

837 

MEYER,  238,  692,  759 
MEYER  AND  EANSOM,  733 
MEYER,  F.  K.,  AND  STICKEL,  103 
MICHAELIS,  114,  305 
MICHEL,  565 
MIGNESCO,  77 
MIGULA,  13,  41,  388 
MIKULICZ,  725 
MILLER,  H.  E.,  608 
MINCHIN  AND  THOMPSON,  1080 
MIQUEL,  37,  1017 
MITCHELL,  200 
MIYJAYIMA,  887 

MOELLER,    116 

MOHLER,  609,  932,  1036,  1042 

MOLDOVAN  AND  DOERR,  366 
MOLTSCHANOFF,  553 

MOMONT,  779 

MONTI,  892 

MOORE,  415,  694 

MORAX,  508 

MORAX  AND  MARIE,  732,  733 

MORGAN,  649,  717 

MORGENROTH,  253 

MORGENROTH  AND  SACHS,  296,  320 

MORISHIMA,  648,  662 

MORO,  228,  608 

MOSER,  926,  928 

MOSER  AND  VON  PlRQUET,  412,  419 
MOSS,  GUTHRIE  AND  GELIEN,  437 

MONTON,  330 
MUCH,  588 
MUELLER,  J.  H.,  32 
Mum  AND  EITCHIE,  756 
MULLER,  619,  398,  1034 
MULLER,  PR.,  388 
MULLER,  OTTO  FRIEDRICII,  2 
MULZER,  852 
MUNSON,  926 


MUNTZ,  27 

MUNTZ  AND  SCHLOSSING,  64 

MYERS,  268,  304 

NANKANISHI,  10,  11,  18,  19,  112 

NAKAYAMA,  428 

NASTJUKOFF,  498 

NEEDHAM,  4 

NEGRI,  902 

NEISSER,   11,   126,  394,  620,  547,  563, 

999 
NEISSER,    BAERMANN,    HALBERSTADTER, 

871 
NEISSER,    KOLLE    AND    WASSERMANN, 

388 

NEISSER  AND  FRIEDMANN,  294 
NEISSER  AND  SACHS,  298,  327 
NEISSER  AND  SHIGA,  708 
NEISSER  AND  WECHSBERG,  252,  297,  393, 

394 

NENKI  AND  SCHEFFER,  22 
NEUFELD,  412,  429,  453,  466,  467,  655 
NEUFELD  AND  HAENDEL,  474,  446 
NEUFELD  AND  HUNE,  338 
NEUFELD  AND  EIMPAU,  337,  467 
NEUFELD  AND  TOPFER,  338 
NEUMANN,  221,  654,  805 
NEUSTAEDTER  AND  THRO,  917 
NEWSHOLM,  574 
NICHOLS,  672 
NICOLAIER,  727 
NICOLL  AND  VINCENT,  842 
NICOLLE,  287,  356,  359,  360,  622,  524, 

858,  937,  939 

NICOLLE,  BLANC  AND  CONSEIL,  943 
NICOLLE  AND  LEBAILLY,  486 

NlKATI   AND   ElETSCH,    836 
NlKOLAYSEN,  553 

NISHI,  881,  885 

NlSHIMURA,    24 

NOCARD,  688,  694,  789,  964 
NOCARD  AND  Eoux,  587,  614,  615 
NOGUCHI,  319,  847,  856,  860,  863,  873, 

881,  887,  905,  906,  1139 
NOGUCHI  AND  KLIGLER,  883 
NOGUCHI  AND  MOORE,  852 
NORRIS,  291,  292,  304,  667 
NORRIS  AND  LARKIN,  964 


1154 


INDEX  OF  AUTHORS 


NORRIS,  PAPPENIIEIMER  AND  FLOURNOY, 
862 

NOTTER   AND   FlRTH,    1035 

NOVY,  759 

NOVY  AND  FRAENKEL,  864 
NOVY  AND  FREER,  78 
NOVY  AND  KNAPP,  847 
NOVY  AND  KNAPP,  861,  866 
NOVY  AND  DE.KRUIFF,  366 
NOVY  AND  MACNEAL,  1077,  1086 
NUTTALL,  249,  250,  277,  292 
NUTTALL  AND  THIERFELDER,  632 

OBERMEIER,  7,  849,  861 
OBERMUELLER,  1042 
OGSTON,  384,  401 
OHNO,  704,   710,  885 
OLITSKY  AND  GATES,  488 
OLITSKY  AND  KLIGLER,  709 
OLMSTEAD,  447 
OMELIANSKI,  56,  65 
OMELTSHENKO,  96 
OPHULS,  988,  744 
OPIE,  595,  500 
OPPENHEIMER,  48,  53 
OSGOOD  AND  LUCAS,  918 
OSTWALD,  47,  146 
OSTERTAG,  693 

OTTO,  355,  359,  360,  880,  878 
OTTOLENGHI,  450 
OVERTON,  238 

PAKE,  198 

PANE,  454,  467 

PAPASOTIRIN,  631 

PAPPENHEIM,  125,  589 

PAPPENHEIMER  AND  MUELLER,  948 

PARK,  106,  269,  270 

PARK  AND  CAREY,  704 

PARK  AND  DUNHAM,  702 

PARK  AND  HOLT,  1033 

PARK  AND  KRUMWIEDE,  600 

PARK  AND  NICOLLE,  740 

PARK  AND  THORNE,  273 

PARK  AND  WILLIAMS,  472,  896 

PARK  AND  ZINGHER,  578,  580 

PARKER,  498 

PARSONS,  490 


PASQUALE,  403,  844 

PASSET,  389,  402 

PASTEUR,  3,  5,  6,  8,  29,  47,  245,  246, 

440,  827,  901,  981 
PASTEUR  AND  CHAMBERLAND,  144 
PASTEUR,     CHAMBERLAND    AND    Eoux, 

245,  780 

PASTEUR  AND  JOURBET,  756 
PAUL,  87,  894 
PEABODY,    DRAPER    AND    DOCHEZ,    209, 

912,  918. 

PEABODY  AND  PRATT,  163 
PEARCE,  253,  550 
PEARCE  AND  EISENBERG,  361 
PEARL,  492 
PEREZ,  726 
PENFOLD,  44 
PERKINS,  723 
PERRONE,  404 
PETERSEN,  348 
PETRI,  617,  1014 
PETRUSCHKY,  416,  639,  649,  654,  961, 

964,  981 
PETROFF,  590 
PETRUSKI,  675 
PETTERSON,  346 
PFAUNDLER,  283,  286 
PFEFFER,  61,  63 
PFEIFFER,  235,  250,  285,  494,  499,  502, 

657,  837,  892 
PFEIFFER  AND  BECK,  501 
PFEIFFER  AND  ISAEFF,  277 
PFEIFFER  AND  KOLLE,  285,  659,  681 
PFEIFFER  AND  NOCHT,  844 
PFEIFFER  AND  UNGERMANN,  709 
PFEIFFER  AND  WASSERMANN,  837 
PFLUGER,  66 
PFUHL,  188,  641 

PlERRALLINI,    331 
V.    PlRQUET,    608 

V.  PlRQUET  AND  SCHICK,  354,  356 

PITT,  14 

PLACE,  929 

PLANT,  867,  984 

PLENCIZ,  2,  3 

PLOTZ,  942 

PLOTZ,  OLITSKY  AND  BAEHR,  941 

POLLENDER,  6 


INDEX  OF  AUTHORS 


1155 


POELS  AND  DHONT,  688 
POOLE,  766 

POOR  AND  STEINHARDT,  905 
PORGES,  14,  287 
POTT,  1035 

POYNTON  AND  PAINE,  422 
PRATT,  656,  671 
PRESCOTT,  631,  1024 
PRATT-JOHNSON,    GILCHRIST,    HAY-MI- 
CHEL, 1130 
PRIGGE,  673 

PRITCHETT  AND  STILLMAN,  500 
PROESCHER,  915,  936 

PROSkAUER    AND    BECK,   32 

VON  PROWAZEK,  859,  943 

PRUDDEN,  649 

PRUDDEN  AND  HODENPYL,  604 

QUIGLEY,  423 
QUINCKE,  1045 

KABINOVITSCH,  617,  625,  941,  992,  1042 

RANSOM,  733 

RANSOME  AND  FULLERTON,  106 

EAVANT  AND  PINOY,  971 

RAVENEL,  613,  779 

VON  RECKLINGSHAUSEN,  7 

EEDTENBACHER,  652 

REED  AND  CARROLL,  688 

SEED,   CARROLL,   AGRAMONTE   AND   LA- 

ZEAR,  875 

EEMLINGER,  905 
EETTGER,  226,  695,  753 
EETTGER  AND  KOSER,  695 
RETTGER  AND  NEWELL,  50 
RICHET  AND  HERICOURT,  248,  396 
EICHARDSON,  78,  654,  656 
EICKETTS,  942,  951,  952,  987 
RlCKETTS  AND  WILDER,  937,  939 
RIDEAL-WALKER,  98 
RIEDER,  79 

RlNDFLEISCH,   7 

RlXFORD   AND  GlLCHRIST,  988 

ROBERTSON,  168,  757 
ROBINSON  AND  EETTGER,  160 
DA  EOCHA-LIMA,  943,  949 

EOEMER  AND  JOSEPH,  916 
EOHNER,    804 


EOGER,   425 
EOMANOWSKY,    127 
EOMER,    741 

EOSENAU,  590,  504,  814 

EOSENAU  AND  ANDERSON,  275,  355,  356, 

570 

EOSENAU,  LUMSDEN  AND  KASTLE,  679 
EOSENAU  AND  McCoY,  1028 
EOSENBACH,  384,  401,  965 

ROSENBERGER,    596 

ROSENOW,  44,  434,  454,  423 
ROSENOW  AND  TOWNE,  423 
ROSENOW  AND  WHEELER,  423 
ROSENTHAL,  708 
Ross  AND  MILNE,  865 
ROST,  621,  624 
ROTHBERGER,  646 
Roux,  11,  78,  180 

ROUX  AND  LlNOISSIER,  991 
ROUX   AND    NOCARD,    792 

Roux  AND  YERSIN,  561,  571,  573 
ROWLAND,  825,  1126 

RUBNER,  26 
RUEDIGER,    428 

RUPPEL,  23,  605 
RUSSELL,  164,  682,  1142 
RUSSELL  AND  FULLER,  1022 

SAATHOF,  315 

SABOURAUD,  996,  999 

SACHAROFF,  872 

SACHS  AND  GEORGI,  329 

SACHS,  238,  250,  253 

SACHSE,  61 

SACQUEPEE,  670,  759 

SAHLI,  607 

SALMON,  828 

SALMON  AND  SMITH,  247,  691 

SANARELLI,  880 

SANFELICE,  992 

SAUERBECK,  350 

SAUL,  94 

SAVAGE,  284 

SCHAEFFER  AND  STEINSCHNEIDER,  552 

SCHAFER,  35 

SCHATTENFROH,  346,  393 
SCHATTENFROH    AND  GRASSBERGER,    1030 
SCHAUDINN,   848 


1156 


INDEX  OF  AUTHORS 


SCHAUDINN   AND  HOFFMANN,   850 

SCHEFFER,   22 

SCHELL   AND   FlSHCER,   593 

SCHELLER,   673 

SCHENK,   993 

SCHERESCHEWSKY,    856 

SCHERING,   109 

SCHEURLEN   AND  SPIRO,  87,  88,  89,  95 

SCHICK,  574 

SCHILD,  631 

SCHIMMELBUSCH,  391 

SCHISTOVITCH,  290 

SCHLOSSING,  64 

SCHLOSSMANN,  107 

SCHNEIDER,  388 

SCHOTTELIUS,  632 

SCHOLTZ,  547 

SCHONE,  674 

ScHOTTMfrLLER,  403,  407,  448,  453,  651, 

689,  965,  1034 
SCHREIBER,  580,  829 

SCHROEDER   AND    COTTON,    800,    1036 
SCHROEDER    AND   DUSCH,   4 
SCHROETER,    67 

SCHUDER,  677,  1032 

SCHUITZLER,    641 

SCHULE,  379 
SCHULLER,  880 
SCHULTZ,  361 

SCHULZE,   4 
SCHUTZ,    414 

SCHUTZE,  253,  290 
SCHWANN,  3,  4,  6 

DE    SCHWEINTZ   AND  DORSET,    24,    603 

SCLAVO,  445,  784 

SEDGWICK  AND  BATCHILDER,  1028 

SEMBAC,  78 

SELLARDS,  936 

SELTER,  487 

SERGENT,  FOLEY  AND  VIALOTTE,  943 

SHARNOSKY,  121 

SHATTOCK,  935 

SHERMAN,  121 

SHIBAJAMA,  692 

SHIRNOFF,  840 

SIIIGA,  700,  702,  710,  716 

SlGNORELLI,   170 
SlLBERSCHMIDT,  641 


SIMON,  LAMAR  AND  BISPHAM,  341 

SIMONDS,  226,  359,  753 

SIMONS,  425 

SIMPSON,  1033 

SIMPSON  AND  HEWLETT,  99 

SMITH,    119,    355,   572,    591,    593,   612, 

613,  919,  1036 
SMITH,  G.,  583 
SMITH,  HERBERT  E.,  1033 
SMITH,  TH.,  29,  30,  39 
SMITH  AND  BROWN,  408 
SMITH,  BROWN  AND  WALKER,  729 
SMITH,     L.,     DRENNAN,     EETTIE     AND 

CAMPBELL,  93 

SMITH,  TH.,  AND  KILBORNE,  246,  693 
SMITH,  TH.,  AND  MOORE,  688 
SMITH  AND  TEN  BROECK,  690,  695    ' 
SOBERNHEIM,  773,  784 
SOBERNHEIM  AND  TOMASCZEWSKI,  851 
SOMMERVILLE,  99 
SOPER,  679 
SOPHIAN,  531 
SORENSEN,  139 
SPALLANAZANI,  4 
SPAGNOLIO,  1097 
SPEAKMAN,  56 
SPENGLER,  607 
SPILKER  AND  GOTTSTEIN,  29 
SPITZER,  851 
SPROUCK,  702 
SPRONK,  572 
STAFSETH,  800 
STANLEY,  491 
STEENSMA,  199 
STEFANSKY,  625 
STEPHEN  AND  FONTHAM,  1090 
STEPHENS,  1131 
STEPHENS  AND  MYERS,  257 
STERN,  291,  664 
STERN  AND  KORTE,  311,  661 
STERNBERG,  38,  388,  406,  448,  440,  564, 

880,  899. 
STTLLMAN,  474 
STICKER,  623 
STOBER,  987 

STOKES  AND  HAECHTEL,  1023 
STOKES,  KYLE  AND  TYTLER,  887 
STOKES  AND  WEGGEFARTH,  1034 


INDEX  OF  AUTHORS 


1157 


STRAUSS,  791 

STRAUSS  AND  GAMALEIA,  604,  615 
STRAUSS,  HIRSCHFELD  AND  LOEWE,  921 
STRAUSS  AND  HUNTOON,  914 
STRELITZ,  568 
STRICHT,  744 

STRONG,  723,  825,  842,  937 
STRONG  AND  MUSGRAVE,  701 
STRONG,  TEAGUE,  CROWELL  AND  BARBER, 
819 

SURMONT,  252 

SURMONT  AND  ARNOULD,  779 
SUZUKI  AND  TAKAKI,  597 
SWIFT,  371,  947 
SWIFT  AND  KINSELLA,  423 

TACKE,  61 

TALAMON,  439 

TAMURA,  23,  24 

TAROZZI,  735 

TAROZZI  AND  SMITH,  167 

TAUTE  AND  HUBER,  1090 

TAYLOR,  KENNETH,  993 

TEAGUE  AND  BARBER,  811 

TEAGUE  AND  DEIBERT,  512 

TEAGUE  AND  STRONG,  813 

TEAGUE  AND  TORREY,  550 

TEAGUE  AND  TRAVIS,  166 

TEN  BROECK,  357 

TERIN,  390 

THAYER,  880 

THOM,  EDMONSON  AND  GILTNER,  743 

THOMAS,  874 

THOMSON  AND  HEWLETT,  220 

TIDSWELL,  675 

TISSIER,  225,  1044 

TISSIER  AND  MARTELLY,  632,  1045 

TIZZONI,  735 

TODD,  394,  708,  709 

TOEPFER,  943,  949 

TOKISHIGE,  992 

DE   TOMA,  593 

TORINI,  53 

TORREY,  223,  228,  550,  553 
TORREY  AND  KAHE,  228,  798 
TOTSUKA,  662 
TOUSSAINT,  245,  780 
TRASK,  929,  1032 


TRILLAT,  106 
TROOMSDORF,  692 
TSIKLIMSKI,  37 
TULLOCH,  522,  525,  737,  378 
TUNNICLIFF,  413,  869 
TURNBULL,  866 

TURRO,  74 

TWORT,  44,  69,  70,  74 

UHLENHUTH,  291,  590 
UHLENHUTH  AND  HUBENER,  698 
UHLENHUTH  AND  MULZER,  858 
ULLMANN,  552 
USCHINSKI,  31,  151,  573 

VAGEDES,  613 

VAILLARD  AND  DOPTER,  708 

VAILLARD  AND  ROUGET,  734 

VAILLARD  AND  VINCENT,  731,  732 

VALNTINE  AND  COOPER,  501 

VALLARI-KADOT,  4 

VAUGHAN,  44,  356 

VAUGHAN  AND  PALMER,  537,  924 

VAUGHAN  AND  WHEELER,  365 

VEDDER,  549 

VEDDER  AND  DUVAL,  702 

VEEDER,  679 

VEILLON,  398 

VEILLON  AND  ZUBER,  752 

VAN  DE  VELDE,  394,  430 

DI  VESTEA  AND  ZAGARI,  901 

VIGNAL,  963 

VlLLEMIN,  586 

VINCENT,  867 

VINCENT  AND  MURATET,  707,  712 

VOGES,  32 

VOTTALER,  728 

WADSWORTH,  118,  445,  450,  457,  467 
WADSWORTH  AND  KIRKBRTDE,  469 
WAGMANN,  1031 
DE  WAELE  AND  SUGG,  412 
WALDEYER,  7 
WALKER,  662 
WARD,  78,  1031 
WASHBURN,  467 

WASSERMANN,  250,  256,  290,  529,  549, 
553,  805,  842 


1158 


INDEX  OF  AUTHORS 


WASSERMANN  AND  BRUCK,  298,  316 
WASSERMANN  AND  CITRON,  350 
WASSERMANN,    NEISSER    AND    BRUCK, 

317 

WASSERMANN  AND  PROSKAUR,  573 
WASSERMANN  AND  SCHUTZE,  291 
WASSERMANN   AND   TAKAKI,   238,   268, 

334,  733 
WASSILIEF,  791 
WATAHIKI,  550 
WATSON,  E.  A.,  1085 
WEEKS,  502 
WEENY,  662    • 
WEGELE,  1046 

WEICHSELBAUM,  515,  560,  398,  440,  720 
WEIGERT,  7,  130,  267 
WEIL,  359,  360,  367,  885 
WEIL  AND  FELIX,  945 
WEISS,  589 

WELCH,  116,  154,  634,  445 
WELCH  AND  BLACHSTEIN,  649 
WELCH  AND  NUTTALL,  211,  752 
WELLS,  120,  356,  368 
WENGON,  1095 
WERNER,  946 

WENICKE,  602,  840,  812,  987 
WERTHEIM,  548 
WESENBERG,  641 
WESTPHAL  AND  UHLENHUT,  623 
WEYL,  29 
WHEELER,  23 
WHERRY,  625,  815 
WHERRY  AND  ERWIN,  518 
WHERRY  AND  OLIVER,  189 
WHIPPLE,  1021 
WICKMAN,  912 
WIDAL,  663 

WlDAL  AND  NOBECOURT,  688 
WILBUR,  745 
WILCKENS,  1032 
WILDE,  724 

WlLLAMON,    33 

WILLIAMS,  33,  501 


WILLIAMS  AND  LOWDEN,  904 

WILLSON,  1023 

WILTSCHOUR,  652 

WILSON,  919 

WILSON  AND  CHOWNING,  951 

WINOGRADSKY,  14,  27,  61,  64,  65 

WINSLOW,  421 

WINSLOW,   KLIGLER   AND   EOTHENBERG. 

647 
WINSLOW  AND  PALMER,  409 

WlNTERNITZ  AND  HlRSCHFELDER,  457 

WLADIMIROFF,  792 

WOLBACH,  890,  936,  950,  951,  988 

WOLBACH  AND  ERNST,  613 

WOLBACH,  SISSON  AND  MEIER,  995 

WOLFF,  350,  635,  760 

WOLF-EISNER,  356,  365,  607 

WOLFF  AND  ISRAEL,  969 

WOLFHUGEL,  80 

WOLLSTEIN,    500,    506,    508,    522,    715, 

930 

WOOD,  113,  128,  450 
WORONIN,  62 

WRIGHT,  127,  221,  181,  183,  681,  899 
WRIGHT,  J.  H.,  967,  968,  1095 
WRIGHT  AND  DOUGLAS,  336,  337 
WRIGHT  AND  MORGAN,  480 
WRIGHT  AND  SEMPLE,  681,  979 
Wu  LIEN  TEH,  817 
WUTZDORFF,  492 
WYNEKOOP,  499 

YERSIN,  807 

ZETTNOW,  861 

ZINSSER,    16,    185,   346,   570,    583,    656, 

705,  955 

ZINSSER  AND  CARY,  626 
ZINSSER  AND  DWYER,  351 
ZINSSER  AND  HOPKINS,  860 
ZINSSER,  PARKER  AND  KUTTNER,  237 
ZINSSER  AND  TSEN,  346 


INDEX  OF  SUBJECTS 


ABBOTT'S  spore  stain,  116 

Absorption  method  in  agglutination,  288 

Acetone  produced  by  the  fermentation 

of  starch,  55  . 
Achorion  Schoenleini,  1001,  1002 

other  species,  -1003 
Acid  and  alkali  production  by  bacteria, 

198 

acids  commonly  found,  199 
Acid-fast  bacteria,  staining  of,  124 

definition  of,  587 
Actinobacillosus,  971 
Actinomyces,  965-971.     See  also  Nocar- 

dia,  964 

animal  inoculation,  970 
appearance  of  in  lesion,  906 
cultivation  of,  968 
definition  of,  962 
diseases  caused  by,  965 
microscopical  examination  and  morph- 
ology, 966,  967 
pathogenicity,  969 
Wright's  method  of  cultivation,  969 
Addison's  disease,  596 
Aerobic  organisms,  obligatory  and  fac- 
ultative, 8 

Aestivo  autumnal  fever,  1107,  1110 
Agglutination,  282 

absorption  method  is,  288 
acid,  284 

agglutinins,  nature  of,  285 
action  on  dead  bacteria,  283 
discovery  of,  251 
major  and  minor,  288 
partial      studied      by      absorption 

method,  288 
production  of,  286 
specificity  of,  288 
theoretical  considerations,  292 


; group,"  288 


Agglutination,   macroscopic,   283 

method    of    bacterial    differentiation, 

282 

microscopic,  282 

practical  application  in  typhoid,  282 
physical  phenomenon  of,  285 
presence  of  electrolyte  necessary  for, 

285 

proagglutinoid  zone,  289 
test,  method  of  performing,  302 
macroscopic,  303 
microscopic,  303 

with  capsulated  organisms,  14,  287 
with  tubercle  bacillus,  302 
Agglutinogen,  287 
Aggressin  theory,  349 
arguments  against,  350 
relation  to  anaphylatoxin,  351 
Air,  bacteria  in,  1010 

in  the  conveyance  of  disease,  1011 
method  of  estimating  the  number  of 

bacteria  in,  1012 
Alcoholic  fermentation,  58 
Alcohols  as  disinfectants,  94,  103 
Alexin   and   sensitizing   antibodies,   277 

281 

discovery  of,  250 
facts  concerning,   295,   296 
Aleuronat  used  for  injecting  rabbits  to 

obtain  leucocytes,  345 
Alkali  production  by  bacteria,  198 
Allantiasis,  740-748 
Allepo  boil,  1095 
Allergy  defined,  353 
Amboceptor,     quantitative     relation     to 

complement,  296 
Amoeba,  1050 
binucleata,  1050 
diploidea,  1050 
proteus,   1050 


1159 


1160 


INDEX  OF  SUBJECTS 


Amoeba,  synopsis  of  genera,  1052 

valk  amphia,  1051 
Amylase,  55 

Anabolic  activities  of  bacteria,  60 
liberation  of  energy,  66 
light  production  by,  66 
nitrogen  fixation,  60 
pigment  formation,  66 
sulphur  production,  67 
Anaphylatoxin,  365 
Anaphylaxis,  352-372 

anaphylatoxin  theories,  365,  366 
antianaphylaxis,  364 
antigen  in,  367 
Arthus  phenomenon,  355 
criteria  for,  356,  357 

Well's  summary  of,  356 
desensitization,  364 

methods  for,  369,  370 
Doerr's  classification,  352,  353 
drug  idiosyncrasies,  371 
hay  fever,  370 

historical  background,  354-356 
observations  by  Arthus,  355 
Hericourt  and  Eichet,  354 
Nicolle,  356 
Otto,  355 

For  tier  and  Kichet,  354 
Eichet,  355 

Eosenau  and  Anderson,  355 
Theobald  Smith,  355 
Vaughan  and  Wheeler,  356 
in  infectious  diseases,  372 
sensitization,  active  methods  for,  358, 

359 

incubation  period  in,  358 
sensitization,  passive,  359 
incubation  period  in,  360 
transmission  by  inheritance,  360 
serum  sickness,  366,  367 
skin  reactions,  368 
delayed,  368 

for   determining  hypersensitiveness 
for  injecting  horse  serum  the- 
rapeutically,  369 
immediate,  368 
site  of  reaction,  361,  362 
symptoms  in  various  animals,  362,  363 


Anaerobic  bacilli,  727-772 

associated    with    traumatic    injuries, 

749-763 

method  of  identifying,  763 
proteolytic  group,  750,  761 
B.  histolyticus,  762 
B.  putrificus,  762 
B.  sporogenes,  761 
saccharolytic  group,  750 
B.  Fallax,  760 
B.  oedematiens,  759 
B.  Welchii,  751 
Vibrion  Septique,  756 
B.  anthraci,  symptomatici,  768-772 
B.  botulinus,  740-748 
B.  Tetanus,  727-740 
Anaerobic  blood  cultures,  214 
Anaerobic    indicator,    methylene    blue, 

187 
Anaerobic  methods  of  cultivation,  179- 

189 

displacement  of  air  by  hydrogen,  182 
indicator  for,  187 

Mclntosh    and    Fildes    palladium   as- 
bestos method,  187 
mechanical  exclusion  of  air,  180 
<  Esmarch's  method,  180 
fluid  media  covered  with  oil,  180 
Liborius  method,  180 
pyrogallol  in,  183 
Eoux's  method,  180 
Wright's  method,  181 
Method  for  combining  exhaustion,  hy- 
drogen replacement  and  oxygen  ab- 
sorption, 185 

Pyrogallic    acid    for    oxygen    absorp- 
tion, 183 
Buchner  tube,  183 

Wright's  modification,  183 
Plate  cultures  of  anaerobes  by  simple 

method,  187 

tissues  in  culture  media,  189 
Anaerobic    organisms,    obligatory    and 

facultative,  28 
methods  of  cultivating,  179 
Andrade   indicator,   147 
Anilin  dyes,  as  staining  agents,  113 
introduction  of,  7 


INDEX  OF  SUBJECTS 


1161 


Anilin  dyes,  Selective  action  of  in  cul- 
ture media,  170,  161 
Animal    experimentation,    autopsies    in, 

204 
bleeding  of  animals  in,  204 

from  the  carotid  artery,  205 

from  the  heart,  205 
methods  of  inoculation,  202,  203 

intraperitoneal,  203 

intravenous,  203 

Kolle  vaccination  method,  204 

subcutaneous,  203 
Animal  holders,  204 
Animal  inoculation,  203 
Antagonism  of  bacteria,  34 

used  in  bacteriotherapy  of  autointoxi- 
cation, 1045 
Anthrax,  773-784 

attenuation  of,  780,  783 
bacteria  resembling,  785 

B.  anthrocoides,  785 

B.  radicosus,  785 

B.  subtilis,   786 
biological  considerations,  778 
cultivation  of,  775-777 
historical  interest  of,  773 
immunity  against,  783 

active  (Pasteur's  work),  783,  784 

passive,  784 
morphology,  774 
pathogenicity,  779 

experimental  inoculation,  780 

in  animals,  780,  781 

in  man,  782 

susceptibility  of  animals,  779 
spore  formation,  778 

resistance  of,  779 
vaccination,  783,  784 
virulence,  780 
Anthrax  symptomatic,  bacillus  of,  768- 

772 

cultivation,  770 
immunity,  771 
morphology,    769 
staining,  769 
toxins,  771 
Antibodies   and   the   substances   giving 

rise  to  them,  249 


Antibodies,    experimentation    and    dis- 
covery of,  249 
agglutinins,  251 
antiferments,  253 
antitoxins,  251 
bacteriolysins,  251 
cytotoxins,  252 
precipitins,  252 
antibodies  sensitizing,  227-281 
Bordet's  views  concerning,  279 
Ehrlich's  views  concerning,  279 
Antiferments,  253 
antilab,  253 
antilactase,   253 
antipepsin,  253 
antisteapsin,  253 
Antiformin   for   concentrating   tubercle 

bacilli  in  sputum,  589 
Antigen,  anaphylactic,  357 
definition  of,  254 
in  the  Wassermann  test,  317,  318 
Antileucocidin     in    staphylococcus    im- 
mune sera,  396 
Antiricin,  250 

Antiseptics,  inhibition  strengths,  104 
Antitoxin,  diphtheria,  269-273 
concentration  of,  272 
immunization  of  horses,  270,  271 
prophylactic  doses,  273 
prophylactic  immunization,  580-582 
regulation  of  by  law,  272 
specific  therapy  in,  579 

dosage,  579 

standardization  of,  271 
therapeutic  doses,  273 
toxin-antitoxin  reaction,  theoretical 

considerations,  255-268 
discovery  of,  250 
tetanus,  274 

immunization  of  horses,  274 
prophylactic  dose,  736 

L-f-  dose,  definition  of,  276 
standardization  of,  275-276 
therapeutic  use  of,  738 

administration  of,  740 
unit  of,  275 
Antivenin,  256 
"  Arnold  »  sterilizer,  83 


1162 


INDEX  OF  SUBJECTS 


Arrhenius    and    Madsen,    theories    con-  j 

cerning  toxin-antitoxin,  264,  265 
Arthus'  phenomenon,  355 
Arthrospores,  not  related  to  true  spores, 

16 

Ash,  bacterial,  24 
Aspergillus,  61,  983 
Attenuated  cultures  in  active  immuniza- 
tion, methods   for,   245 

with  anthrax,  780 
Autoclave,  85 

table  of  relation  between  temperature 

and  pressure,  86 
Autointoxication,  gastro-intestinal,  1044 

Metchnikoff  's      bacteriotherapy      in, 

1045 

Autolysins,  300 
Autopsies  of  experimental  animals,  204 

BABES,-EENST,  granules,  discussion  of, 

11 
special  stains  for,  126 

Babesia  bigeminum,  1131 

Bacilli,  9 
genus  of,  41 

Bacillus  abortus  (Bang),  799-801 
isolation  from  mlik,  800 
lesions  produced  by,  in  guinea  pigs, 

800 
See  also  B.  abortus  equi,  693 

Bacillus  abortus  equi,  693 
See  also  cattle  abortion,  799 

Bacillus  acidi  lactici,  639 

Bacillus  acidophilus  in  the  normal  in- 
testinal tract,  225 
description  of,  228 

Bacillus  acidophilus  aerogenes,  descrip- 
tion of,  228 

Bacillus    actinomycetum    comitans,    oc- 
currence of  in  mycosis,  970 

Bacillus  aerogenes  capsulatus,  752 
Sec  B.  Welchii 

Bacillus  anthracis,   773.     See  also  An- 
thrax 
See  also  Symptomatic  anthrax,  768 

Bacillus  anthracis  symptomatic!,  768 

Bacillus  anthrocoides,  785 

Bacillus  avisepticus,  827 


Bacillus     avisepticus,     Pasteur 's     early 

work  with,  828 
Bacillus  Belloncnis,  759 
Bacillus  bifidus  in  the  normal  intestinal 

tract,  225,  227 
description  of,  228 
Bacillus  botulinus,  740-748 
antitoxin,  747 

clinical  manifestations,  746 
cultivation  of,  741 
isolation  of,  742 
morphology,    741 
occurrence  of,  744-746 
pathology  of,  744 
prevention  of,  748 
specific  therapy,  747 
staining  of,  741 
'toxin,  742-744 

not  produced  in  the  tissues  of  mam- 
mals, 744 

thermolability  of,   743 
transmission  of,  744 

relation  to  limberneck  of  chickens, 

746 

types  of,  744,  748 
Bacillus   bovis    morbificans    (Basenau), 

688 
B.  bronchisepticus  in  canine  distemper, 

798 

Bacillus  bulgaricus,  1046 
Bacillus  butryicus,  617 
Bacillus  chauvaci,  759.     See  also  Symp- 
tomatic anthrax 
Bacillus  cloacae,  641 
Bacillus  coli  communior,  637 
Avery  type,  637 
Malia  type,  637 

Bacillus  coli  communis,  628-637 
cultivation  of,  629 
distribution  of,  631 
immunization  with,  635 

normal  agglutinins  for,  636 
morphology  of,  628 
occurrence  of  in  the  intestinal  tract, 

225 

occurrence  of  in  the  normal  nose,  221 
occurrence  of  in  water,  1023 
presumptive  test,  1024 


INDEX  OF  SUBJECTS 


1163 


Bacillus  coli  communis,  occurrence  of  in 
water,  table  for  identification,  1026 
pathogenicity  of,  632-634 
septicemia  due  to,  633 
poisonous  products  of,  635 
varieties  of,  636 
WinkePs  disease  due  to,  633 
Bacillus  dentifricans,  60 
Bacillus  diphtherias,  561-582.     See  also 

Diphtheria  bacillus 
Bacillus  enteritidis  (Gaertner),  687 
meat  poisoning  due  to,  698 

clinical  picture  in,  699 
Bacillus  fallax,   760 
Bacillus  fecalis  alkaligenes,  639 
Bacillus  granulobacter  peetinovarum,  55 
Bacillus  histolyticus,  762 
Bacillus  Hoffmanni,  582 

occurrence   of  in  the  normal  mouth, 

219 

Bacillus  icteroides,  688 
Bacillus  influenzas,  482.    See  also  Influ- 
enza 

Bacillus  lactis  serogenes,  637-639 
in  the  normal  intestine,  225 
in      Metchnikoff  's      Bacteriotherapy, 

1044 

in  the  mouth,  219 
in  the  nose,  221 

Bacillus  leprae,  620.     See  also  Leprosy 
Bacillus     Mallei,     787-794.       See     also 

Glanders 

Bacillus  maximus  baccalis  in   the   nor- 
mal mouth,  220 
Bacillus  melitensis,   795-797 
animal  pathogenicity,  796 
cultivation  of,  796 
disease  caused  by,  796 
epidemiology,  797 
immunity,   797 
morphology,  795 

Bacillus  Morseele  (van  Ermengen),  688 
Bacillus  of  meat  poisoning,  686,  698 
Bacillus  mucosus  capsulatus,  720 
cultivation,  721 

general  characteristics  of  group,  722 
morphology,  720 


Bacillus  mucosus  capsulatus,  pathogen- 
icity, 723 
staining,  722 
Bacillus  oedematiens,  759 
antitoxin,  760 
method  of  identifying,  763 
pathogenicity,  760 
toxin,  760 

Bacillus  oedematiens  maligni  II,  759 
Bacillus  of  cattle  abortion,  799-801 
isolation  from  milk,  800 
lesions  produced  by,  in  guinea  pigs, 

800 
Bacillus  of  chicken  cholera,  827 

Pasteur's  early  work  with,  828 
Bacillus  of  fowl  typhoid,  694 
Bacillus  of  Ghon  and  Sachs,  756 
Bacillus  of  guinea  pig  pneumonia,  801- 

802 

Bacillus  of  swine  plague,  829 
Bacillus  of  whooping  cough,  504-508 
Bacillus  ozsenae,  726 
Bacillus     perfringens,  752.       See     also 

B.  Welchii 

Bacillus  pestis,  807.    See  also  Plague 
Bacillus  pestis  caviae,  692 
Bacillus     phlegmoiiis      emphysematosa?, 

752.    See  also  B.  Welchii 
Bacillus  prodigiosus,  quantitative  chem- 
ical analysis  of,  22 
Bacillus  proteus,  640,  641 

agglutination  of  in  typhus  serum,  642, 

945 

pathogenicity  of,  641 
Bacillus  psittacosis,  694 
Bacillus  putrificus  in  the  normal  intes- 
tinal tract,  225 
description  of,  229,  762 
Bacillus  pullorum,  695 
Bacillus  pyocyaneus,  802-806 

antitoxic  substances  produced  by,  806 
cultivation,  803 

pigment  production  by,  803,  804 
immunization,  805 
morphology,  802 
pathogenicity,  804 
staining,  802 
toxins,  805 


1164 


INDEX  OF  SUBJECTS 


Bacillus  radicicola,  62 
Bacillus  radicosus,  785 
Bacillus  Ehinoscleroma,  724 

pathogenicity  of,  725 
Bacillus  sanguinarium,  694 
Bacillus  smegmatis,  618-619 
Bacillus  sporogenes,  761 
Bacillus  subtilis,  786 
Bacillus  suisepticus,  829 
Bacillus  tetanus,  727-740.    See  also  Te- 
tanus 

Bacillus  III  of  von  Hibler,  756 
Bacillus  tuberculosis,  586.    See  also  Tu- 
berculosis 
Bacillus   typhi    abdominal) s,    643.      Sec 

also  Typhoid  bacillus 
Bacillus  typhi  murium,  691 
Bacillus    typhosus,    643-685.      See    also 

Typhoid  bacillus 
Bacillus  Welchii,  751-756 

agglutinin  production,  753 

antitoxin,  755 

cultivation,  752-753 
fermentations  of,  753 

hemolysin   production,   753 

importance  of  in  intestinal  autointox- 
ication, 1044 

importance    of   in   the    normal    intes- 
tinal flora,  226 

isolation  of,  755,  756 

method  of  identifying,  763 

morphology  of,  752 

pathogenicity  of,  753 

spore  formation,  752 

staining  of,  752 

toxin  production,  754 
Bacillus  Xerosis,  584 
Bacteria  (see  also  Bacterial  cell) 

acid  and  alkali  formation  by,  198 

anabolic  or  synthetic  activities  of,  60 

antagonism  of,  34 

chemical  agents  injurious  to,  86 

classification,  39 

based  on  organs  of  motility,  16 
by  Migula,  41 

counting  of,  194-195 
in  milk,  1038 

cultivation  of,  172 


Bacteria,  cultivation  of,  by  anaerobic 
methods,  179 

degenerative  forms,  20 

denitrifying,  59 

destruction  of,  76 

differentiation  by  fermentation,  54 

enzymes  produced  by,  54,  201 

gas  formation,  196 

Gram  negative,  123 

Gram  positive,  123 

in  air,  1010 

in  milk,  1027 

in  the  intestinal  tract,  2 
at  different  ages,  224,  225 
in  relation  to  diet,  226,  227 

in  the  normal  mouth,  217-220 

in  the  normal  nose,  220-222 

in  the  tissues,  223 

in  water,  1020 

katobolic  activities  of,  46 

microscopic   study  of,   111 

nitrifying,  64 

nutrition  of,  27 

parasitic,  33 

pathogenic,  231 

physical  agents  injurious  to,  76 
relation  of  to  moisture,  39 
relation  of  to  pressure,  39 
relation  of  to  temperature,  36 

relationship  to  other  plants,  40 

reproduction  of,  19 

saprophytic,  33 

size  of,  9 

staining  of,  113 

sulphur,  67 

symbiosis,  34 

thermal  death  points  of,  38 

variations  in  form  of,  20 
Bacteriaemia,  definition  of,  233 
Bacteriaceae,   definition  of,   41 
Bacterial  cell,  ash  in,  24 

Babes-Ernst  granules  in,  11 

capsule  in,  12 

chemical  constituents  of,  21 
fats  in,  24 
proteins  in,  22,  25 

membrane  of,  12 

metachromatic  granules,  11 


INDEX  OF  SUBJECTS 


1165 


Bacterial  cell,  morphology  of,  10 

motility  of,  14 

nucleus  in,  10 

osmotic  properties  of,  25 

plasmolysis,  25 

plasmoptysis,  25 

specific  gravity  of,  26 

spores  in,  16 

water,  22 

Bacteriocidal    and    bacteriolytic     tests. 
307-311 

bacteriolysins,  discovery  of,  251 

determination  of  the  bacteriolytic 
power  of  serum  against  a  known 
organism  in  vivo,  308 

identification  of  a  microorganism  by 
observing  its  susceptibility  to  lysis 
in  a  known  immune  serum,  309 

reaction  in  test  tubes,  310 
' '  Bacteriophage ' '  phenomenon,  69 

Bordet  and  Cuica's  work,  72 

early  work  of  Twort,  70 

discovery  of  lytic  principle  by  d'Her- 
elle  in  filtrates  from  the  stools  of 
dysentery  and  typhoid  convales- 
cents, 71 

Bacterium  aceti,  57 

Bacterium  clostridium  pasteurianum,  61 
Bacterium,  genus,  41 
Bacterium  pasteurianum,  57 
Bacterium   pneumonias,    720.     See   also 

Bacillus  mucosus  capsulatus 
Bacterium  tularesne,  826 
Bail's  aggressin  theory,   349 

arguments  against,  350 

relation  to  anaphylatoxin,  351 
Balantidium  Coli,  1137 
Bang  Bacillus  abortus  bovis,  799 
Barber   pipette   method   of    isolating   a 

single  organism,  178 
Baumgarten's  stain  for  differentiating 

between  the  tubercle  bacillus  and  the 

bacillus  of  leprosy,  125 
Beggiatoa,  genus,  42 
Beggiatoaceae,  definition  of,  42 
Berkefeld  filter,  144 
Biological  activities  of  bacteria,  45 
Black  death,  807 


Blackleg,   769.     See  also  Symptomatic 

anthrax 

Black  water  fever,  1128 
Blastomycetes,     relationship     to     other 

plants,  40 

Blastomycosis,  984-987 
blastomyces  hominus,  985 
classification  of,  987 
cultivation,  986 
isolation,  986 

microscopic   examination,   985,    986 
morphology,  986 
pathogenicity,   987 
staining,  986 

Bleaching  powder  as  a  disinfectant,  89 
Bleeding  of  animals,  204 
from  the  heart,  204 
from  the  carotid  artery,  205 
from  the  external  jugular  vein,  205 
Blood  cultures,  technique  of  taking,  212- 

215 

method  of  obtaining  blood,  212 
media  for,  213,  214 
from  typhoid  patients,  214 
anaerobic,  214 
Blood  examination  for  protozoa,   1140- 

1143 

Blood  grouping,   313-315 
Jansky's  classification,  313 
Moss'  classification,  313 
"universal  donor,"  314 
Blood,  method  of  obtaining,  204,  205 
defibrinated,    method     of     obtaining, 

205 
enriching   substance   to   be   added   to 

media,  169 
ether   added   as   a  preservative,    205, 

169 

BodonidsB,  1076 

Boiling  as  a  method  of  sterilization,  83 
Bordet-Gengou  bacillus,  504-508 
antibody  production  by,  508 
cultivation  of,  506 
epidemiology,  504-505 
morphology  of,  505 
pathogenicity  of,  507 
staining  of,  506 
Bordet-Gengou  phenomenon,  298 


1166 


INDEX  OF  SUBJECTS 


Bordet's   interpretation   of  toxin   anti- 
toxin reaction,  265 
Botulismus,  740-748.     See  also  Bacillus 

botulinus 
Brilliant  green  as  a  disinfectant,  97 

in  culture  media,  163 
Brill's  disease,  934.     Sea  also  Typhus 

fever 

Brownian  movement,  14 
Buboes,  809,  813 
Buchner  tube  for  anaerobic  cultivation, 

183 
" Buffers"  in  culture  media,  definition 

of,   138 

Burning,  as  a  method  of  sterilizing,  82 
Butter  bacillus,  617 
Butter,  bacteria  in,  1039-1042 

Tubercle  bacilli  in,  1042 
Butyl  alcohol  produced  by  fermentation 

of  starch,  55 

CADAVERINE,  51 

Capsules,  description  of,  12,  13 

relation  to  virulence,  14 

special  stains  for,  116 
Carbolic  acid,  95 

coefficient  of,  98 

determination,  of,  101 
Carbon    dioxide   produced   by   bacteria, 

196 

Carbon  in  the  nutrition  of  bacteria,  27 
Carrel    smear     method     of     examining 

wounds,  764 
Carriers  as  a  source  of  disease,  377 

in  diphtheria,  575-577 

in  dysentery  amoebic,  1062 

in  dysentery  bacillary,  714 

in  malaria,  1117,  1123 

in  meningitis,  542 

m  typhoid,  669-676 
Cellulose,  12,  24,  56 
Carcomonadidse,  1075 

Bodonidae,  1076 

Trypanosomidae,  1076 
Cercomonas  homini,  1055 
Chagas  (Schizotrypanum  curzi),  1091 
Chancroid,  Ducrey  bacillus  in,  512 
Charbon,  773.    See  also  Anthrax 


Charbon  symtomatique,   768.     See  also 

Symptomatic  anthrax 
Chaulmoogra  oil,  in  treating  lepers,  625 
Cheese,  hard  and  soft,  bacteria  in,  1043 
Chemical  constituents  of  the  bacterial 
cell,  21 

bacterial  ash,  24 

lipoidal  constituents,   24 

nature  of,  the  antigenic  proteins,  25 

quantitative     chemical     analysis     of 
mass  cultures,  22 

types  of  bacterial  protein,  22,  23 
Chemotaxis  in  phogocytosis,  332 
Chicken  cholera,  bacillus  of,  827-829 
Chicken  pox,   distinct  from  small  pox, 

894 

China  blue  indicator,  148 
Chitin  in  bacteria,  24 
Chlamidozoa,  1139 
Chlamydor  bacteriaceae,  definition  of,  42 

description  of,  961 
Chloramine  T,  93 

Chloride  of  lime  as  a  disinfectant,  89 
Cholera,  Asiatic,  spirillum  of,  831 

animal  pathogenicity,  836 

biological  considerations,  835 

cholera-red  reaction,  833 

cultivation  of,  832-834 

Dieudonne's  selective  medium  for, 
833,  166 

disease  caused  by,  in  man,  835 

epidemiology,   837 

endemic  foci,   838,   840 

immunization,  841 

isolation  of  from  feces,  834 

isolation  of  from  water,  834 

morphology,  831 

prophylactic  vaccination  of,  841-843 

staining,  831 

toxin,  837 
Cholera-like  spirilla,  845 

Spirillum  Deneke,  845 

Spirillum  Finkler  Prior,  845 

Spirillum  Massaua,  844 

Spirillum  Metchnikovi,  844 
Chorea,    streptococcus    viridans    associ- 
ated with,  423 
Chromobacteria,  66 


INDEX  OF  SUBJECTS 


1167 


Chromophytosis,  1008 
Cladothrix  asteroides,  964 

defined,  692 

genus,  42 
Classification  of  bacteria,  41 

according  to  flagella  (Massea),  16 

according  to  Migula,  41 

of  the  more  important  pathogenic 
bacteria  according  to  Gram 's  stain, 
123 

Clinic  relation  to  bacteriology,  373 
Coccaese,  definition  of,  41 
Cocci,  description  of,  9 
Coccidiodal  granuloma,  987 

coccidiodes  immunitis,  988 
Cohni  streptothrix  israeli,  965 
Cold,  the  common,  436-438 

filtrable  virus  in,  discussion  of,  437 

epidemiological  considerations,  488 
Colon  bacillus,  group  of,  627-640 

cultivation  of,  629 

definition  of,  637 

differentiation  of,  by  sugar  fermen- 
tations, 637 

distribution  of,  631 

immunization  with,  635 

in  the  intestinal  tract,  appearance  of, 
225 

in  the  normal  nose,  221 

in  water,  1023 

presumptive  test,  1024 
table  for  identification,  1026 

pathogenicity,  632-634 

poisonous  products  of,  635 

varieties  of,  636 

Colony  fishing,  technique  of,  177 
Colony  study,  193 

colony  counting,  194 
Color  standards  for  colorimetric  method 

of    titrating   media,    preparation    of, 

140 

Comma  bacillus,  831.     See  also  Cholera 
Complement   deviation    (Neisser-Wechs- 

berg  phenomenon),  297 
Complement,  facts  concerning,  295 

effect  of  concentration,  295 

filtration  of,  295 

inactivation  of,  295 


Complement,  inhibition  of,  295 

separation  into  fractions,  296 
Complement    fixation    by    precipitates, 
296 

determinatoin  of  antibodies  by;  315, 
316 

determination  of  antigen  by,  325 

for  protein  differentiation,  327 

in  glanders,  794 

in  the  Wassermann  test,  317 

in  tuberculosis,  608 
Cowpox  relation  to  small  pox,  894 
Crenothrix,  genus,  42 
Cristispira,  847 
Crytococcus  Gilchristi,  985 
Culex   fatigans   in   the  transmission  of 

Dengue  fever,  931 

Culture  media,  133-171.    See  also  Media 
Cytorryctes  variolse,  892 
Cytotoxins,  discovery  of,  252 

nephrotoxins,  253 

DARIN'S  solution,  production  of,  90 

Dansyz,  effect,  265 

Dansyz  type,  692 

Dark-field  in  the  demonstration  of  spiro- 

chseta  pallida,   853 
Delousing,  953 

crude    creosote    oil    in    personal   pro- 
phylaxis, 955 

bath  house,  arrangement  of,  955 

gaseous  disinfection,  956 
formaldehyde,  956 
sulphur  dioxide,  956 
hydrocyanic  gas,  956 

heat,  aplication  of,  956,  957 
Degeneration  forms  of  bacteria,  20 

variations  not  degeneration  forms,  20 
Delhi  boil,  1095 
Dengue  fever,  931-932 

transmission   by  culex   fatirans,   931, 
932 

clinical  symptoms,  931 
Denitrifying  bacteria,  59 
Denitrification,  59 
Dermatophytes,  type   of  fungi   causing 

ringworm,  995-1009 


1168 


INDEX  OF  SUBJECTS 


Dermentor   Venustus  -in.  the    transmis- 
sion    of    Kocky    Mountain     Spotted 
fever,  951 
Desensitization,  364 

methods  for,  369,  370 
Destruction  of  bacteria,  76 
by  chemical  agents,  86 
gaseous,  105 
in  solution,  86 
inorganic,  87 
organic,  94 

by  physical  agents,  76 
drying,  76 
light,  77 
electricity,  79 
heat,  79 

Diastase.    See  Amylase 
Dichloramine  T,  93 
Dientamoeba  fragilis,  1072 
Diet,  effect  on  intestinal  flora,  224,  226 
Diphtheria  bacillus,  561-582 
antitoxin,  269-273 

concentration  of,  272 
immunization  of  horses,  270,  271 
prophylactic  doses,  273 
prophylactic  immunization,  580-582 
regulation  of  by  law,  272 
specific   therapy,   578-580 

dosage  in,  579 
standardization  of,  271 
therapeutic  doses,  273 
toxin-antitoxin  reaction,  theoretical 

considerations,  255-268 
unit,   definition  of,  271 
biological  characteristics,  564 
carrier  problem  in,  575-577 
cultivation  of,  565 

Loeffler's  medium,  565 
diagnosis,  567 

diphtheria-like  bacilli,  582-584 
bacillus  Hoffmanni,  582 
bacillus  Xerosis,  584 
diphtheria  bacilli,  584-585 

differentiation  by  sugar  fermenta- 
tions, 585 
discovery  of,  561 
epidemiology  of,  574-577 
carriers  in,  575-577 


Diphtheria  bacillus,  isolation,  566 
morphology,  562 
pathogenicity,  567-570 
causes  of  death,  569 
for  animals,  570 
' '  pseudomembranes, ' '  567 
resistance  of,  564 
Schick  reaction,  577 
staining,  562 

Neisser  stain,   563 
Toluidin  blue,  564 
thermal  death  point,  564 
toxin,  571-574 

in  antitoxin  reaction,  theoretical 

considerations,  225-268 
chemical  and  physical  properties 

of,  573 

Ehrlich's  analysis  of,  257-263 
deterioration  of,  258 
expitoxoid  or  toxon,  definition 

of,  261 

Limes  death  or  L-f-  dose,  defin- 
ition of,  260 

Limes  zero  in  L0  dose,  defini- 
tion of,  259 
method   of  partial  absorption, 

262 

standardization,  258 
toxoid,  259 
unit   of,   257 

method  of  production,  571 
thermolability,  572 
virulence   determination 
Diphtheroids,  description  of,  584,  585 

in  the  normal  nose,  221 
Diplococcus  crassus,  535 
Diplococcus  gonorrhoeas,  547.     See  also 

Gonococcus 
Diplococcus  lanceolatus,  438.     See  also 

Pneumococcus 
Diplococcus  mucosus,  536 
Diplococcus  pneumoniae,  438.     See  also 

Pneumococcus 

Discomyces,  964.     See  also  Nocardiae 
bovis,  965 

in  madura  foot,  972 
Disinfectants,  86-109 


INDEX  OF  SUBJECTS 


1169 


Disinfectants,  bactericidal  strengths  of, 

105 

gaseous  for  fumigation,  105 
ehloriii,  106 
formaldehyde,  106 
hydrocyanic   acid   gas   for   rodents, 

109 

oxygen,   106 
sulphur  dioxide,  106 
inhibition  strengths  of,   104 
inorganic,  87 

efficiency  of  acids,  salts  and  bases 
proportional  to  degree  of  disso- 
ciation, 87 
halogens,  89 

chloride    of    lime,    or    bleaching 

powder,  89 
eusol,  93 

terchloride  of  iodin,  93 
tincture  of  iodin,  93 
iodoform,  93 
oxydizing  agents,  93 

peroxide  of  hydrogen,  93 
potassium  permanganate,  94 
organic,  94 
alcohols,  94 
carbolic  acid,  95 
essential  oils,  96 
flavine  dyes,  96 
formaldehyde,  95 
formalin,  95 
lysol,  95 
tricresol,   95 

triphenylmethane  dyes,  97 
brilliant  green,  97 
malacite  green,  97 
testing  efficiency  of,  methods  for,  97 

98 

Eideal  Walker,  98 
U.  S.  Public  Health  Service,  99 
Distemper,  canine,  798 
Doderlein's    bacillus,    occurrence    of   in 

vagina,  230 
Dourine,  1084 

Drying,  resistance  of  bacteria  to,  76 
Ducrey  bacillus,  510-513 

cultivation  in  rabbit's  blood,   167 
etiological  factor  in  chancroid,  512 


Ducrey  bacillus,  isolation  of,  511 

pathogenicity,  513 
Drug  idiosyncrasies,  371,  352 
DTN  M250,  meaning  of,  257 
Dyes,  anilin,  as  staining  agents,  113 

selective  action  of  in  media,  170,  161 
Dysentery,  amosbic  (Entamoeba  histoly- 

tica),  1054-1062 
autopsy  findings,  1055 
carriers  in,  1062 
complications  following,  1055 
cyst  formation,  1060 
degenerative  forms,  1060 
diagnosis,  1056 
distribution,  1055 
epidemiology  of,  1063 
multiplication,  1057 
observation  of  fresh  specimen,  1058 
staining  methods,  1057 
treatment  in,  1062 
Dysentery,  bacillary,  700-717 
carriers  in,  714 
clinical  description  of,  711 
epidemiology  of,  711-715 

transmission  of,  713,  714 
fermentations  of  the  bacilli  causing, 
707 

compared  with  typhoid-paratyphoid 

group,  718 

general  characteristics  of  bacilli  caus- 
ing, 706 

historical  survey,  700-704 
immunization  with  dysentery  bacilli, 

710 

poisonous  products  of  dysentery  ba- 
cilli, 708 

action  on  animals,  709 
prevention  of,  711 
prophylactic  vaccination,  716 
resistance  of  dysentery  bacilli,  709 
serum  treatment  of,  717 
thermal  death  point,  707 
types  of  bacilli  causing,  704-707 

Flexner,  704,  707 

Shiga,  700,  704,  707 

Strong,  704,  707 

"Y,"  704,  707 


1170 


INDEX  OF  SUBJECTS 


EHRLICH'S  analysis  of  diphtheria  anti- 
toxin,  257-263 

Ehrlich  'a  side  chain  theory,  216-268 
Electricity  in   the   destruction   of   bac- 
teria, 79 

Empyema,  following  pneumonia,  465 
Endocarditis,  gonococcus  in,  553 

streptococcus  viridans  in,  422,  423 
Encephalitis  lethargica,  919 

clinical  picture  in,  920 

etiology,  920,  921 

discovery  of  globoid  bodies  in,  921 

relation  to  influenza,  920 

spinal  fluid  in,  920 
Endoenzymes,  49 
Endolimax,  nana,  1068 
Endotoxins,  235 

probably  protein,  24 
Energy  of  bacteria,  source  of,  29,  30 

derived  from  oxidation  of  ammonia, 
65 

derived  from  oxidation   of  hydrogen 

sulphide,  68 
Entamceba,  1051 
Entamoeba  Butschlii,  1068  (iodamceba 

Butschlii) 
Entamceba  Coli,  1063 

cyst  formation,  1065 

infection,  experimental  with,  1066 

morphology,  1064 

multiplication,  1065 
Entamoeba  gingavalis,  1066 

morphology  of,  1067 

cyst  formation  of,  1067 

histolytica,  1054-1062 

(etiological  factor  in  amosbic  dysen- 
tery, see  Dysentery  amo3bic) 

nana,  see  Endolimax  nana,  1068 

tetragena  africana,  1060 
Enteritidis,  bacillus  of  (Gaertner),  687 

meat  poisoning  due  to,  698 

clinical  picture  in,  699 
Enterococcus,  399 

in  the   normal  intestinal  tract,   225. 

227 
Enzymes,  bacterial,  as  katalysers,  47 

amylase  or  diastase,  55 
method  of  testing  for,  212 


Enzymes,  cellulase,  56 

gelase,  56 

invertase,  56 

method  of  testing  for,  202 

lab,  53 

lactase,  56 

lactic  acid  fermentation,  56 

lipase,  53 

maltase,  56 

of  fermentation,  54 

oxydases,  57 

proteolytic,  48 

method  of  testing  for,  49,  201,  202 

xymase,  in  alcoholic  fermentation,  58 
Epidemiology    of    infectious     diseases, 

377-383 

van  Ermengen's  stain  for  flagella,  119 
Erysipelas,  streptococcus  in,  416 

treatment    with    leucocyte     extracts, 

433 

Erythrasma,  1009 
Esmarch  roll  tubes  for  obtaining  single 

colonies,  177 

Essential  oils  as  disinfectants,  96 
Eusol,  93 
Exudates,  examination  of,  207,  208 

FARCY,  790 

Fats  in  bacterial  cell,  24 

Fat-splitting  enzymes  produced  by  bac- 
teria, 53 

Favus,  1001 

Ferments.     See  Enzymes,  47 

Fermentation,   54 

as  a  source  of  energy,  30 

alcoholic,  58 

ammoniacal,  52 

caused  by  both  yeasts  and  bacteria, 

46 

due  to  bacterial  enzymes,  47 
lactic  acid,  56 

Feces,  examination  of,  211 

isolation  of  tubercle  bacilli  from,  212 
isolation  of  typhoid  bacilli  from,  653 
kinds  of  organisms  present,  211 
isolation  of  anaerobes  from,  by  rab- 
bit inoculation,  211 
number  of  bacteria  in,  211 


INDEX  OF  SUBJECTS 


1171 


Filters,  types  of: 
Berkefeld,  144 
Chamberlaud,  144 

cleaning  and  sterilization  of  filters, 

145 
FHtrable  virus,  general   considerations, 

889-890 
diseases  caused  by,  Wolbach  's  chart 

of,  891 
in  connection  with  the  common  cold, 

437 

in  influenza,  486-488 
Flagella,  15 

arrangement  and  structure,  15 
staining  of,  118 

Flavine  dyes  as  disinfectants,  96 
Flexner  bacillus  in  bacillary  dysentery, 

704,  707 
Foot-and-mouth  disease,  932-933 

due  to  a  filtrable  virus,  933 
Formaldehyde,  disinfectant  action  of,  95 
methods   for   producing    for    fumiga- 
tion, 107,  108 
Breslau  apparatus,  107 
v.  Brunn  apparatus,  118 
Lentz  apparatus  using  glycerin  to 

prevent  polynecrization,  107 
"lime"  method,  108 
potassium    permanganate     method, 

109 

Trillat  autoclave,  109 
Formalin,  95 

bactericidal  strength  of,  104 
inhibition   strength   of,    103 
methods    of  producing   formaldehyde 
from,  107.    See  also  Formaldehyde. 
Fowl  typhoid,  694 
Frambo3sia  tropica,  870 
Friedlander 's  bacillus,  720 

occurrence   of  in  the  normal  mouth, 

219 

occurence  of  in  the  normal  nose,  221 
pathogenicity  of,  723 
Fumigation,  105-110 
chlorine  in,  106 
formaldehyd,  107-109 
hydrocyanic  gas  for  rodents,  109 
oxygen,  106 


Fumigation,  ozone,  106 

sulphur  dioxide,  106 
Fungi,  pathogenic,  973-984 

ascomycetes,  979 
yeastes,  980 

classification  of,  975 

hyphomycetes      (fungi      imperfecti), 
983-1009 

morphological   definitions,   974 

phycomycetes,  978 
mucor,  978,  979 
Furunculosis,  391 
Fusiform    bacilli    in   Vincent 's   angina, 

867 
Fusiform  bacilli  other  than  those  found 

in  Vincent's  angina,  869 

GABBET'S  stain  for  the  tubercle  bacil- 
lus, 124 
Gaertner  bacillus,  687 

meat  poisoning  due  to,  698 

clinical  picture  in,  699 
Gas    bacillus,    751-756.      See    also    B. 

Welchii 
Gas  formation  by  bacteria,   196 

analysis  of,  197 

determined    by    Smith    fermentation 
tube,  196 

hydrogen  sulphide,  197 
Gelase,  56 
Genitalia,  examination  of  lesions  on,  216 

syphilitic,  216 

chancroid,   216 
Gentian  violet,  selective  action  in  media, 

170 
Geryk  pump  for  producing  a  vacuum, 

908 

Giardia  intestinalis,  1074 
Giemsa's  stain,  127 
Glanders  bacillus,  787-794 

biological  considerations,  789 

complement  fixation,  794 

cultivation,  787-788 

diagnosis  of,  791 
Strauss  test  in,  791 

immunity,   794 

morphology,  787 

pathogenicity,  789 


1172 


INDEX  OF  SUBJECTS 


Glanders     bacillus,     pathogenicity,     in 

horses,  789-791 
in  man,  789,  790 
nodule  formation  in  the  chronic 

form  of  the  disease,  791 
staining,  587 
toxins  of,  791 

mallein,  diagnositic,  use  of,  792,  793 
mallein,  preparation  of,  792 

Glassware,  method  of  cleaning  and  ster- 
ilizing, 133 

Globoid  bodies  in  poliomyelitis,  915 

Globulin    in    spinal    fluids,    method    of 
testing  for,  209 

Glycerin  in  culture  media,  169 

for  growth  of  tubercle  bacillus,  592 

Gonococcus,  547-559 
antibodies  to,  553 
complement  fixation  with,  553 
cultivation  of,  548-550 
endocarditis  due  to,  553 
fermentation  reactions,  535 
identification  of,  551,  552 

by  sugar  fermentations,  551 
morphology  of,  547 
ophthalmia  due  to,  555 
pathogenicity,  552 
prophylaxis,  559 
rheumatism  due  to,  553 
resistance,  552 
thermal  death  point,  552 
types  of,  550 
vaccine  therapy,  554 
vulvovaginitis  due  to,  556 

Gram-negative  bacteria,  important  path- 
ogenic list  of,  123 

Gram-negative   cocci,   table   of  fermen- 
tations, 535 

Gram-positive  bacteria,  important  path- 
ogenic list  of,  123 

Gram's  stain,  121 

Gram-Weigert  method  of  staining  Gram 
positive  organisms  in  tissues,  130 

Group  agglutination,  282.    See  also  Ag- 
glutination. 

Grubor-Widal  reaction,  302 

H^MOPKILIC  group  of  organisms,  482 


Haffkine's  virus,  825,  826 

Halogens  as  disinfectants,  89,  104,  103 

' '  Hanging  block  method ' '  for  studying 
living  bacteria,  112 

" Hanging  drop"  method  for  studying 
living  bacteria,  111 

Halteridium,  1099-1101 

Hay  fever,  370 

Heat,  in  the  destruction  of  bacteria,  79 
dry  and  moist,  comparison  of,  80 
low     efficiency     of     ' '  superheated ' ' 

steam,  82 
live  steam,  83 
steam  under  pressure,  85 

Helber  counting  chamber  for  bacteria, 
195 

Hemorrhagic-Septicaemia  group,  807-829 
bacillus  of  chicken  cholera,  827 
bacillus  of  plague-like  diseases  in  ro- 
dents, 826 
bacillus  Pestis,  807 
bacillus  of  swine  plague,  829 

Hemolysin,  280 
discovery  of,   252 
immune,  discovery  of,  279 

specificity  of,  299 
production  by  staphylococcus,  393 
production   by   streptococcus    hemoly- 
ticus,  426 

Hemolytic  tests,  311 

Hemoproteus  columbae,   1099-1101 

Hemosporida,  1098 

d  'Herelle  's   ' '  Bacteriophage ' '   phenom- 
enon, 70,  71 

Hermann's  stain  for  the  tubercle  bacil- 
lus,  126 

Higher  bacteria  for  trychomycetes,  701 

Hiss'  capsule  stain,  116 

Hiss'  leucocytic  extract,  344-346 

Histamine,  52 

liberated  by  abnormal  proteolytic  ac- 
tivity in  the  intestinal  tract,  227 
produced  in  bacterial  cultures,  237 

Hoffmann  bacillus,  582 

Hog-cholera,  bacillus  of,  691 

Hot  air  sterilization,  82 

Huntoon'g  capsule  stain,  117 


INDEX  OF  SUBJECTS 


1173 


Hydrocyanic   acid   gas,    destruction   of 

rodents  by  fumigation  with,  109 
Hydrogen  in  the  nutrition  of  bacteria, 

31 

Hydrogen  ion  determination,  138-142 
by  colorimetric  method,  138-142 
Ph,  meaning  of,  138,  138 
preparation  of  color  standards  for, 

141 
indicators    for    various    ranges    of 

Ph,  140,  141 
actual  steps  in,  142 
' '  buffer, ; '  definition  of,  138 
comparator,  for  reading,  142 
Hydrogen    sulphide    produced    by   bac- 
teria, determination  of,  197 
Hydrophobia,  900.     See  also  Eabies 
Hypersusceptibility,  definition  of,  352 

Doerr's  classification,  352,  353 
Hypomycetes,  983 
in  relationship  to  other  plants,  40 

IMMUNITY,  defined,  240,  241 
natural,  241 
racial,  242 
species,  241 
acquired,  243 
active,  244 

produced    with    attenuated    cul- 
tures, 245 
produced    with    sublethal    doses, 

246 

produced  with  dead  bacteria,  246 
produced  with  bacterial  products, 

247 

passive,   247,   248 
antitoxic  sera,  247 
antibacterial  sera,  247 
Impetigo  contagiosum,  392 
Ictero-haemorrhagic  fever,  885.   See  also 

Weil's  disease. 
Incubators,   190 

thermo  regulators,  191 
Indicators,  theory  of,  146 

for  hydrogen  ion  determination,  140, 

141 

phenol  red  or  phenolsulphonephtha- 
lein,  141 


Indicators  for  hydrogen  ion  determina- 
tion, brom-thymol  blue,  141 
cresol  red,  141 

forjaddition  to  culture  media,  147 
Andrade,  147 
china  blue,  148 
litmus,  147 
neutral  red,  148 

Indol  production  by  bacteria,  199 
method  of  testing  for,  199 

Vanillin  test,  199 

Infantile  paralysis,  912.  See  also  Polio- 
myelitis 

Infantile  splenomegaly,  1095 
Infection,  definition  of,  230 
factors  determining,  232,  233 
path  of,  232 
Infectious  diseases,  transmission  of  by 

direct  contact,  380 
by  insects,  380 
in  intestinal  group,  379 
through  respiratory  channels,  378 
Influenza  bacillus,  482-501 
biology  of,  498 
clinical  picture,  482,  483 
cultivation  of,  495-498 
preservation  of,  497 
epidemiology,  488 

characteristics    of    the    epidemics, 

489 
history  of  the  last  War  epidemics, 

493,  494 

secondary  and  tertiary  waves,  char- 
acteristics of,  492 
transmission,  490 
etiology  of,  484 

arguments   for   and   against   Influ- 
enza bacillus,  485,  486 
filtrable  virus  theory,  486-488 
in  trachoma,  500,  501 
isolation  of,  495 

Avery  sodium  oleate  agar,  496 
morphology,  494 

occurrence   of   in   inter-epidemic   per- 
iods, 499 

pathogenicity  for  animals,  501 
stains,  494 
toxin  formation,  499 


1174 


INDEX  OF  SUBJECTS 


Influenza  bacillus,  varieties  of,  500 

Infusoria,  1137 

Inhibition  strength,  of  various  antisep- 
tics, 103 

Inoculation  of  media,  172 
type  of  wire  for,  173 
technique    of    transferring    cultures, 
175 

Intestinal  tract,  bacterial  flora  of,  223- 

228 

influence  of  age,  224,  225 
influence  of  diet  on,  224,  226 
influence  of  B.  Welchii  in,  226,  227 

Intravital    method    of    studying    living 
bacteria,  112 

Invertase,  56 

lodamojba  Butschlii,  1069 

Iodine,  tincture  of,  as  a  disinfectant,  93 

lodoform,  93 

Isoagglutinins  in  human  blood,  313-315 
Jansky's  classification,  313 
Moss  classification,  313 
"universal  donor,"  314 

Isolation  of  bacteria  in  pure  culture  by, 

174 
pour  plates,  175 

technique  of,  175 
colony  fishing,  technique  of,  177 
streak  plates,  technique  of,  178 

Isolysins,  299,  300 

JAUNDICE,  epidemic,  885-888 

clinical  description,  885-886 

etiology,      spirochaeta      icterohaemor- 

rhagiae,  886 
cultivation  of,  886,  887 

prevention,  888 

serum  treatment,  888 

transmission,  887 
Jenner's  stain,  127 
Jensen's  modification  of  Gram's  stain, 

122 

KALA-AZAR,  1093 

Katabolic  activities  of  bacteria,  46 

Kefyr,  1631 

Kernig  sign  in  meningitis,  527 


Klebs-Loeffler  bacillus,  561.     See  also 

Diphtheria  bacillus 
Koch-Weeks  bacillus,  502 
Kolle  vaccination  method  for  B.  pestis, 

204 

Koumys,  1031 
Kreatoxismus,  51 


L-(-  DOSE,  diphtheria  toxin,  260 

L0  dose,  diphtheria  toxin,  259 

Labferment,  53 

Lactase,  56 

Lactic   acid   bacilli  in  bacteriotherapy, 

1044 

Lactic  acid  fermentation,  56 
Lambia    intestinalis,    1074.      /See    also 

Gicirdia  intestinalis,   1074 
Landry's  paralysis,  912,  915 
Latency  of  bacteria  in  tissues  and  cir- 
culation, 223 
Leischmania,  1093 
Leischmania  braziliansis,  1097 
Leischmania  donovani,  1093 

animal  pathogenicity,  1095 

morphology,  1093 
Leischmania  infantum,    1095 
Leischmania  nilotica,  1097 
Leischmania  tropica,  1095 
Leprolin,  624 
Leprosy,  bacillus  of,  620-625 

cultivation  of,  621 

discovery  of,  620 

distribution  of,  622,  623 

morphology  of,  620 

pathogenicity,  622 

clinical  varieties  of,  623 
contagiousness  of,  624 
reaction  of  lepers  to  tuberculin,  625 
therapeutic  use  of  Chaulmogra  oil, 

625 
Leprosy,  rat,  625,  626 

acid-fast  bacilli  in,  626 

cultivation  of,  626 
Leptothrix,  962,  963 

in  the  normal  mouth,  220 
Leptospira,    class    of   spiral    organisms, 

849 


INDEX  OF  SUBJECTS 


1175 


Leptospira    icterohaemorrhagiae,    causa-  i 
tive  agent  of  yellow  fever,  881-883  ! 

description  of,  882 

immunization  with,  884 

medium  for  cultivation  of,  882 

relation  to  Weil's  disease,  883 
Leucocidin,  Nakayama  's  method  of  test- 
ing for,  429 

production  by  staphylococcus,  394 

production  by  streptococcus,  428 

specificity  of,  238 

Leucocytic  substances   (Hiss),  effect  of 
injections  of,   344-346 

method  of  obtaining,  345 
Lice,  953 

baths      for      individual      proctection 
against,  955  j 

crude  creosote  oil  for  prophylaxis,  955  j 

habits  of,  953,  954 

heat,  in  delousing,  956,  957 

in  the  transmission  of  typhus  fever,  I 

937 
Riekettsia  bodies   in,   943 

varieties  of,  953 

Light  in  the  destruction  of  bacteria,  77 
Light  production  by  bacteria,  66 
Limber  neck  in  chickens,  relation  to  bo- 

tulismus,  746 
Lipase,    enzyme   produced   by   bacteria, 

53 
Lipoidal   constituents   of    the   bacterial 

cell,  24 

Lipovaccine,  typhoid,  481,  683 
Litmus  indicator,  147 
Lockjaw,  727.  See  also  Tetanus 
Loeffler's  medium  for  diphtheria,  565 
.Loeffler's  method  for  staining  bacteria 

in  tissues,  129 

Loeffler's  stain  for  flagella,  119 
Ludwig's  angina,  417 
Lysins,  278,  279 
Lysol,  95,  104 

Lytic  agents,  transmissible  in  series,  69 
Lyssa,  900-909.     See  also  Rabies 

MACROPHAGE,  331 
Madura  foot,  971,  972 
Madurella  in  madura  foot,  972 


Malachite  green  as  a  disinfectant,  97 

in  culture  media,  163 
Malaria,   1102-1131 

carrier  state  in,  1117,  1123 
clinical  description  of,  1115-1117 
cultivation  of  in  vitro,  1118 
description  of  the  mosquito,  1118 
development  of  the  larva,  1119 
incubation  period  in,  1121 
life  history,  1121 

development  of  human  malarial  para- 
sites in  the  mosquito,  1117 
epidemiology,   1122 
carriers  in,  1123 
prevention    of    mosquito    breeding, 

1122 

quinine  prophylaxis,  1125 
screening,  1122 
treatment,  1124 

examination  of  fresh  blood,  1114 
geographical   distribution,   1103 
history  of,  1102 
immunity  in,  1127 
life  cycle  of  parasite,  1106 
other  malarial  parasites,  1131 
pathology,  1126 

plasmodia  finer  structure  of,  1112 
plasmodium  falliparum  causing  aes- 

tivo  autumnal  fever,  1111 
staining  of,  1114 

plasmodium  malariae  causing  quar- 
tan fever,  1110 
staining  of,  1114 
plasmodium   vivax,   causing  tertian 

fever,   1107-1110 

susceptibility  of  negroes  to,  1105 
transmission  by  various  species,  ano- 
pheles mosquitoes,  1118 
treatment  of,  1129 
Malignant  edema,  bacillus  of.     See  Vi- 

brion  septique;  B.  sporogenes 
Mallei n,  791 

diagnostic  use  of,  792 
preparation  of,  791 
Mallory  's  eosin  methylene-blue  stain  for 

bacteria  in  tissues,  131 
Malta  fever,  795.   See  also  B.  melitensis 
Maltase,  56 


1176 


INDEX  OF  SUBJECTS 


Marmosets,  species  of  monkey,  suscep- 
tible to  Leptospira  icterohaemorrha 
giae,  881 

Mastigophora  (class  of  protozoa),  1075 
Measles,  922-926 
virus  in  blood,  922 

virus  in  nasal  and  pharyngeal  wash- 
ings, 923 

epidemiology,  923-926 
infectious  period,  925 
incubation  period,  926 
pneumonias  following,  925 
prevention  of,  925 

Meat  poisoning  due  to  B.  botulinus,  740 
Meat  poisoning  due  to  Gaertner  baccil- 

lus,  698 
Media,  133-171 
anaerobic  tissue  tubes  for  spirochsete 

cultivation,  167 
cooked  meat  medium  for  cultivating 

anaerobes,  168 
dyes,  selective  action  in  media,  170, 

also  see  161,  162 

enriching  substances  added  to,  168 
ascitic,  hydrocele  or  pleura!  fluids, 

169 

blood,  169 

method  of  obtaining,  169 
preservation  of  blood  by  adding 

ether,  169 
Formulas   of   media   in    general    use, 

148-156 

agar,  hormone  or  vitamine,  149 
lactose  litmus   (Wurtz),  153 
meat  extract,  148 
meat  infusion,  148 
sodium   oleate  for   the   influenza 

bacillus,  156 
starch   (Vedder),  154 
trypager,  153 

broth,  calcium  carbonate,  156 
for     streptococcus     agglutination 

(Avery),  150 
glycerine,  150 
hormone  or  vitamine,  149 
meat  extract,  148 
meat  infusion,  148 
potato,  155 


Media,  forrnulae  in  general  use,  sugar- 
free  broth,  150 
"chocolate"  media,  158 
Dorset  egg  medium,  154 
Dunham 's  solution,   151 
Gelatin,  meat  extract,  151 
gelatine,  meat  infusion,  152 
glycerine  egg  (lubenau),  154 
hormone  media,  149 
Loeffler's  medium,  156 
medium   for   cultivating   nitrifying 

bacteria,  65 
milk  media,  156 
nitrate  broth,  151 
pepton  salt  solution  (Dunham),  151 
serum      water      for     fermentation 

(Hiss),   157 
synthetic  media  for  tubercle  bacilli, 

155 

Uschinsky's  protein  free,  151 
Welch 's  modification  of  Guanieri  's 

medium,  154 

General  methods  of  preparing,  133 
clearing  with  egg,  143 
filtration,  143 
ingredients,  135 

meat  extract  and  meat  infusion 

defined,  135 
slanting  of,  146 
sterilization  of,  by  144 
autoclaving,   144 
filtration    through    Berkefeld    or 

Chamberland  filters,  144 
fractional  method,  144 
titration  of,  136 

colorimetric  method,  138 
actual  steps  in,   142 
"buffer,"  definition  of,  138 
color     standards,     preparation 

of,  140 
indicators   for   various    ranges 

of  Ph,  141 

Ph,  meaning  of,  138,  139 
phenolphthalein,  old  method,  136 
Indicators  added  to,  147-148 
Rabbits'   blood   for   Ducrey  bacillus, 
167 


INDEX  OF  SUBJECTS 


1177 


Media,  formulae  in  general  use,  special  for 
colon-typhoid  differentiation,  158 
Barsickow's  medium,  164 
bile  medium,  163 
brilliant  green  agar  (Krumwiede), 

161 
brilliant  green  eosin  agar  (Teague 

and  Clurman),  162 
Conrad  i-Drigalski,   158 
Endo's,  159 

Kendall's  modification  of,  160 
Robinson's  and  Rettger's  modifi- 
cation, 160 

Jackson's  lactose  bile  medium,  163 
lead  acetate  agar  for  Partyphoid  A 

and  B   differentiation,    163 
lead     acetate     added     to     Russell 

double  sugar,  165 
Mackonkey's  bile  salt,  164 
malachite    green    broth     (Peabody 

and  Pratt),   163 
Neutral  red  medium,   164 
Eussell  double  sugar,  164 

with  lead  acetate,  165 
special,  for  isolation  of  cholera  spir- 

alla,  166 
Aronson's,  166 
Dieudonne,  166 
Teague  and  Travis,  167 
Meningitis,  acute  primary,  514 

due  to  meningococcus  or  pneumococ- 

cus,  514 
epidemic,  514 
secondary,  514 
Meningococcus,  514,  546 
agglutination,  523 

agglutinin  absorption,  522 

diagnostic,  543,  544 

method  of  producing  agglutinating 

serum  for  laboratory  use,  523 
technique  of,  524 
animal  pathogen icity,  526 
carrier  determination,  542 

diagnostic    agglutination,    543,    544  j 

media  for,  542 

West   tube    for   taking   nasopharn-  ! 

geal  swabs,  542 
cultivation  of,  516 


Meningococcus,  cultivation  of,  influence 

of  CO2  on,  518 
media  for   fermentation   reactions, 

520 

optimum  Ph,  518 
optimum  temperature,  518 
special  media  for,  519,  520 
diagnosis  of,  527 

differentiation  from  other  Gram  neg- 
ative cocci,  533 
epidemiology,  536-546 
carriers,  539 
cure  of,  545 

determination  of,  542-544 
rate  of,  540,  541 
seasonal  prevalence,  537 
susceptibility  of  negroes,  537 
susceptibility,  variability  of,  539 
fermentation  reactions  of,  535 

medium  for,  520 
morphology  of,  515 
resistance  of,  520 
septicaemia  due  to,  527,  529 
serum  therapy  in,  529,  533 
serum,  administration  of,  530 
dosage  in,  531 
intravenous  injections,  532 
effect  of,  532,  533 
standardization    by    agglutination, 

530 

spinal  fluid,  method  of  obtaining,  527 
spinal  fluid,  examination  of,  528,  529 
staining  of,  515 
toxic  products,  521 
types  of,  522 
virulence,  546 

Metchnikoff  's  bacteriotherapy,  1044 
Metchnikoff 's    work     on    phagocytosis, 

330,  334 

Methylene-blue,  anaerobic   indicator,  187 
Micrococci.     See  Staphlyococci,  384 
Micrococcus,  genus,  41 
Micrococcus    candicans    in    the    normal 

mouth,  218 

Micrococcus  catarrhalis,  553,  559-560 
differentiation    from    gonococcus   and 

meningococcus,  559 
fermentation  reactions  of,  535 


1178 


INDEX  OF  SUBJECTS 


Micrococcus  catarrhalis,   occurrence   of 

in  the  normal  mouth,  218 
Micrococcus  crassus,  535 
Micrococcus  flavus,  534 

in  the  normal  mouth,  218 
Micrococcus  intracellularis  meniiigitidis, 

504.     See  also  Meningoeoccus 
Micrococcus   melitensis,   795.     See  also 

B.  melitensis 

Micrococcus  pharyngis  siccus,   535 
occurrence   of   in  the  normal  mouth, 

218 
Micrococcus  tetragenous,  398 

present  in  the  normal  mouth,  218 
Microphage,  331 

Microscopic  study  of  bacteria,  111 
in  fixed  preparations,  112 

method  of  making  a  smear,  113 
see  staining,  113 
in  living  state, 

by  "hanging  drop"  method,  111 
by  "hanging  block"  method,  111 
by  intravital  method  of  Nakanishi, 

112 

Microsiphonales,  961 
Microspira,  genus,  42 
Microsporon,     1003.      See    also    Micro- 
sporon  furfur  and  Microsporon  min- 
utissum,  1009 

Milk,  alcoholic  fermentation  in,  1031 
bacteria  in,  1027 
"bitter,"  1031 

butyric  acid  formation  in,   1030 
"certified,"  1029 
counting  bacteria  in,  1038 
dairy  inspection,  1037 
pasteurization,  1037 
regulation  concerning,  1040 
relation  to  infectious  disease,  1031 
anthrax,  occurrence  of,  in,  1035 
cholera  traced  to,  1033 
diphtheria  traced  to,   1033 
foot-and-mouth     disease     transmitted 

by,   1035 

infantile  diarrhea  attributed  to,  1034 
scarlet  fever  traced  to,  1033 
streptococci  in,  1034 


Milk,  streptococci  in,  epidemic  of  sore 

throat  caused  by,  1034 
tuberculosis     transmitted    by,     1035- 

1037 

tuberculin  test  for  cattle,  1037 
species  of  bacteria  in,  1029 
source  of  bacteria  in,  1027 
souring  of,  1029 
Kefyr,   1031 
Koumys,   1031 
sterilization  of,  1037 
Milzbrand,  773.     See  also  Anthrax 
Moeller's  spore  stain,  116 
Moisture,    importance    of    in    bacterial 

growth,  39 

Molds,  in  the  normal  nose,  221 
occurrence  of,  982 
pencillium  glaucum,  983 
aspergilli,  983 
sterignocystis,  983 
Monilia  albicans,  989 
Monilia  psilosis,  991 

relation  to  sprue,  991 
Morax-Axenfeld  bacillus,  508 
Morgan    bacilli    in    infantile    diarrhea, 

717 

Motility  of  bacteria,  14 
conditions  favoring,  15 
organs  of,  14 
true      motility      distinguished      from 

Brownian  movement,  14 
Mount  Desert  bacillus  in  bacillary  dys- 
entery  (Park),  704,  707 
Mouth,  bacteria  in,  217,  218,  219 
Much's  granules  in  tubercle  bacilli,  588 
Mucor,  979 
Mumps,  930 

incubation  period,  930 
Muriseptus,  bacillus  of,  503 
Mutation,  bacterial,  43 

problem  of,  in  connection  with  strep- 
tococcus, 434 

Mycetoma  or  Madura  foot,  971,  972 
Myeoderma  dermatitis,  985 

NAGANA,  1081 

Nakayoma's    method    for    testing    for 
leucocidin,  429 


INDEX  OF  SUBJECTS 


1179 


Negri  bodies  in  rabies,  902 

cultivation  of,  by  Noguchi,  905,  906 

diagnosis  of  rabies  by,  903 

van  Gieson's  method  for  rapid  dem- 
onstration, 903,  904 

significance  of,  904,  905 

staining  in  tissues,  902 
Neisser's  stain  for  polar  bodies,  126 
Neisser-Wechsberg  phenomenon,  297 
Nephrotoxin,   253 
Neutral  red  indicator,  148 
Nitrifying  bacteria,  64 
Nitrite  formation  by  bacteria,  200 

method  of  testing  for,  200 
Nitrogen  fixation  by  bacteria,  60 
Nitrogen  in  the  nutrition  of  bacteria,  31 
Nitromonas  or  nitrosomonas,  64 
Nocardia,  963-965 

in  rat-bite  fever,  965 

in  Madura  foot,  972 

minutissimum,  1009 

Noguchi  'a  butyric  acid  test  for  globu- 
lin, 209 

Noguchi  'a  globoid  bodies  in  poliomye- 
litis, 915,  916 
Noguchi 's  method  of  cultivating  spiro- 

chaeta  pallida,  856 
Non-specific  protein  therapy,  347 

clinical  picture  following,  348 
Nose,  normal  bacterial  flora,  220 

filtering  action  of  nasal  mucous  mem- 
brane, 221 

percentage   findings    of    various    bac- 
teria, 221 

Novy  jar  in  anaerobic  cultivation,  185 
Nutrition   of   bacteria,   elements   neces- 
sary, 27 

carbon,  27 

oxygen,  28 

nitrogen,  31 

hydrogen,  31 

salts,  31 

substances   of   unknown    composition, 
32 

vitamines,  33 

OIDIUM  albicans,  989 
Oidium  Hektoenii,  985 


Oidium  lactis,  in  cheese,  1042 

therapeutic  use  of,  1045 
Oospira,  964 

Ophthalmia  due  to  gonococcus,  555 
Opsouic  index,  341 
Opsouic  test,  339 

in  judging  effect  of  vaccine  therapy, 

344 
Simon,  Lamar  and  Bispham  'a  method, 

341 
Wright's  technique  of,  339 

method  of  obtaining  bacterial  emul- 
sion, 339 
method  of  obtaining  blood  serum, 

339 

method  of  obtaining  leucocytes,  340 
Opsonins,   336 

multiplicity  of,  337 
Neuf eld's  and  Bimpau's  work  on,  337 
relation    to    antibodies    and    comple- 
ment, 338,  339 
specificity,  337 
thermolabilrty  of,  338 
Wright's  work  on,  336,  337 
Osteomyelitis,  caused  by  staphylococcus, 

392 

caused  by  streptococcus,  416 
Osmotic  properties  of  bacterial  cell,  25 
Oxydases,  57 
Oxydising  agents  as   disinfectants,   93, 

103,  104 

Oxygen,  in  the  nutrition  of  bacteria,  28 
bactericidal  action  of,  106 
partial  tension  for  the  growth  of  bac- 
teria, 189 

Oysters,  examination  of,  1047 
Ozaenae,  bacillus  of,  726 

Perez  bacillus  of  ozaense,  726 

PASTEURIZATION  of  milk,  1037 

Paltauf 's  modification  of  Gram's  stain, 

121 
Pappenheim-Saathof  stain,   115 

for  gonococcus  smears,  548 
Pappenheim's  stain  for  differentiating 

between  the  tubercle  bacillus  and  the 

smegma  bacillus,  125 


1180 


INDEX  OF  SUBJECTS 


Pappenheimer 's    method    for    staining 
Gram  positive  bacteria  in  tissues,  131 
Parasites,  defined,  33,  231 
facultative  parasites,  34 

not  necessarily  pathogenic,  231,  232 
Paratyphoid-enteritidis  group,  686-699 
differential      considerations,      within 

group,  669 
fermentation    reactions,    chart    of, 

690.     See  also  718 
differentiation   of   from   B.  coli  and 

typhoid,  687 

general  survey  of,  687-689 
Paratyphoid  A,  defined,  689 

pathogenicity  for  man,  696 
Paratyphoid    B,   heterologous    group, 

698 

pathogenicity  for  animals,  691-695 
B.  abortus  equi,  693 

distinguished  from  B.  abortus 

bovis,  693,  799 

B.    enteritidis     (Gaertner    bacil- 
lus), 687,  698 
B.   of  fowl  typhoid    (B.  sangui- 

narium),  694 
B.  Pestis  caviae,  692 
B.  psittacosis,   694 
B.  pullorum,  695 
B.  typhi  murium,  691 
Danysz  type,  692 
hog  cholera,  691 
pathogenicity  in  man,  697 

meat  poisoning  due  to  Gaertner  ba- 
cillus (B.  enteritidis),  698 
clinical  picture  in,  699 
typhoid-like  fever  due  to,  697 
Paratyphoid  C,  688 
preventive  measures,  699 
Paronychia,  391 
Penicillium,  983 

in  cheese,  1043 
Perez  bacillus  of  Ozaena,  726 
Peripneumonia  of  cattle,  10 
Perlsucht,  tuberculosis  of  cattle,  612 
Peroxide  of  hydrogen,  as  a  disinfectant, 

93,  104 

Pertussis  bacillus,  504-508 
antibody  production,  508 


Pertussis  bacillus,  cultivation  of,  506 

epidemiology,  504,  505 

morphology  of,  505 

pathogenicity,  507 

staining  of,  506 
Petroff's  method  of  isolating  tubercle 

bacilli  from  sputum,  591 
Petruschky's  scheme  of  the  higher  bac- 
teria, S62 

Pfeiffer  bacillus,  482.    See  Influenza. 
Pfeiffer  phenomenon,  251 
Ph,  meaning  of,  138 
Phagocytic  index,  in  opsonic  tests,  341 
Phagocytosis,  330-335 

chemotaxis,  332 

" fixed"  cells,  331 

of  dead  and  living  matter,  332 

theoretical  considerations,  334,  335 
Metchnikoff's  work,  330,  334 

variations  in  phagocytic  response,  333 

variations    of   susceptibility    of    bac- 
teria to,  333 

"  wandering  "  cells,  331 
macrophages,  331 
microphages,  331 
Phenol  production  by  bacteria,  200 

method  of  testing  for,  200 
Phenol   red   indicator   in   hydrogen   ion 

determination,  141 
Phenol-sulphon-phthalein,  141 
Phenolphthalein     method     of    titrating 

media,  136 

Pharynx,  normal  bacterial  flora,  217 
Phragmidiothrix,  genus,  42 
Phycomycetes,     group     of     pathogenic 

fungi,  978 

Physical  properties  of  bacteria,  26 
Pigment  formation,  by  bacteria,  66 

condition  necessary  for,  67 

red  pigment  of  thiobacteria,  68 
Piroplasmidae  (Faunea),  1131 

clinical  observations,  1133 

morphology,  1132 

transmission  by  ticks,  1133 

life  history  of  the  tick,  1133-1135 
Pityriasis  versicolor,  1008 
Plague  bacillus,  807-825 

animal  pathogenicity,  815 


INDEX  OF  SUBJECTS 


1181 


Plague    bacillus,    autopsy    findings    in 

rats,  822 

bacteriological  diagnosis,  820 
biological  considerations,  810 
cultivation,  809 

on   salt   agar   to   obtain   involution 

forms,  810 

disease,  caused  by,  in  man,  813 
incubation  period,  813 
pneumonic  form,  814 
transmission  by  flea  bite  or  inhala- 
tion, 813 
epidemiology,  814 

pneumonic    form,    transmission    of, 

819 

prevalence  of,  815 
rats  in  the  transmission  of,  816 

due  to  flea,  817 
immunization,  812 
isolation,  809 
morphology,  808 
prevention  of,  820 

circumvention  of  a  focus,  823 
rat  destruction  in,  820 
rat  proofing  in,  823 
staining,  808 
toxin  formation,   812 
vaccination,  824-826 

Haffkine's  virus,  825 
Plague-like  disease  of  rodents,  826 
Planococcus,  genus,  41 
Planocarcina,  genus,  41 
Plasmodia,  finer  structure  of,  1112 
Plasmodium  falciparum,  1111,  1106 
Plasmodium  malariae,  1110,  1106 
Plasmodium  vivax,  1106,  1107-1110 

minuta,  1131 
Plasmolysis,  12,  25 
Plasmoptysis,  26 
Pleuropneumonia  of  rabbits,  bacillus  of, 

503 
Plotz  bacillus,  association   with  typhus 

fever,  941,  942,  944 
Pneumonobacillus,    720.      Rcc    also    B. 

mucosus    capsulatus 
Pneumococcus,  438-481 

antibody  formation,  466,  467 


Pneumococcus,  as  the  cause  of  primary 
meningitis,  514 

bile  test  for  identification,  453 

blood  cultures  for,  464 

capsule  formation,  440,  441 

complications    following    pneumonia, 
465 

cultivation   of,   442-446 

description  of  on  blood  agar,  445 
on  inulin  •  serum  water,  445 

epidemiology  of,  472-480 

primary  pneumonias,  473-478 
autoinfection,  discussion  of,  474, 

475 

prevention  of,  477 
susceptibility  factor  in,  475,  476 
secondary  pneumonias,  478 
prevention  of,  479 

etiological  factor  in  lobar  pneumonia, 
434 

hemolysin  production  by,  445 

inulin  fermentation  by,  445,  453 

immunity,  466 

immunization,  active,  480 

immunization,  passive,  467 
serum  production,  468 
serum  treatment,  470-472 
standardization  of  serum,  467,  470 

in  the  normal  mouth,  218 

in  the  normal  nose,  221 

morphology  of,  440-442 

mucosus  Type  II    (streptococcus  mu- 
cosus), 448 

pathogenicity  of,  455 

intratracheal   method    of   injecting 
rabbits,  457,  459 

protection  tests,  469,  470 

resistance  to  disinfectants,  450 

staining  of,  442 
in  tissues,  442 
capsules,  442 

serum  treatment,  470-472 

thermal  death  point,  450 

types  of,  446-448 

subgroups  of  type  II,  447,  448 
incidence  in  lobar  pneumonia,  463 
incidence  in  normal  persons,  459 

typing  from  sputum,  460 


1182 


INDEX  OF  SUBJECTS 


Pneumococcus,     typing     from     sputum, 

mouse  injection,  461 
agglutination  of  peritoneal  exu- 

date,  462 
precipitins  in  peritoneal  exudate, 

463 

by  protection  experiments,  463 
Avery  method,  462 
urine,  precipitins  in,  method  of  deter- 
mining, 463 
viability  of,  449 
virulence  of,  455 

method  of  increasing,  456 
Poisons,  bacterial,  234-239 
endotoxins,  235 
mode  of  action  of,  238 
toxins,  definition  of,  234 
"x"  substances,  237 
Polar  bodies,  11 

Poliomyelitis,  acute  anterior,  912-918 
clinical  picture,  913 
epidemiology,  917 
carrier  state,  918 
fly  transmission  discussed,  918 
etiology,  914,  915 

globoid  bodies  in,  915,  916 
filtrability  of  virus,  915 
immunity,  916,  917 
spinal  fluid  in,  913 
transmission  to  monkeys,  914 
transmission  to  rabbits,  916 
Polymastigna  (order  of  protozoa),  1073 
Pompholyx,  1007 
Phosphorescence,  produced  by  bacteria, 

66 
Potassium    permanganate,    disinfectant 

action,  94 

Pour  plates,  technique  of,  175 
Precipitation,  289 
precipitins,  290 

absence  of  in  normal  sera,  292 
discovery  of,  252 
specificity  of,  290 
tests,  304 

preparation  of  bacterial  filtrates 
and  protein  solutions  for,  306 
theoretical  considerations,  292 


Precipitation,  precipitins,  production  of 

precipitating  sera,  304,  305 
Pressure,  effect  of,  on  bacteria,  39 
Protein  cleavage  by  bacteria,  46 
Proteins,    as    antigens,    254.      See    also 
Antigen  of  the  bacterial  cells,  22, 
23,  25 

racemized,  not  antigenic,  357 
Proteolytic  enzymes,  48 

method  of  testing  for,  49 
Proteosoma  praecox,    1101 
Proteus  group  of  bacilli,  640 

agglutination   in  typhus   serum,   642, 

945 
occurrence   of   in  the   normal  mouth, 

219 

pathogenicity,  641 
Protomonadina,   1075 

Cercomonadidae,  1075 
Protozoa,  pathogenic,  1048 
classification,  1048 
technique   of  blood   examination  for, 

1140,  1143 

Pseudomones,  genus,  42 
Pseudodiphtheria  bacillus,  582 
Pseudoinfluenza  bacillus,  501 
Pseudo-membranes  in  diphtheria,  567 
Pseudo-tuberculosis  in  guinea  pigs  due 

to  B.  pestis  carviae,  692 
Ptomaines,  50 

chemical  constitution  of,  51 
discovery  of,  234 
Public  health  management  of  venereal 

diseases,  556 

Purpura  hemorrhagica,  clinical  differen- 
tiation from  typhus  fever,  935 
Pus,  examination  of,  207 
Putrefaction,  as  a  source  of  energy,  30 

definition  of,  50 
Putrescine,  51 
Pyemia,  defined,  233 
Pyocyanin,    pigment    produced    by    B. 

pyomaneus,  804 
Pyocyanolysin,  806 
Pyrogallic  acid  in  anaerobic  cultivation, 

183 

Pyorrhea,  1066 
amoeba  in,  1066 


INDEX  OF  SUBJECTS 


1183 


QUARTAN  fever,  1106 

clinical  description,  1115-1117 

Quarter-evil,   769.     See  also   Symptom- 
atic anthrax 

BABBIT  inoculation  for  isolation  of  anae- 
robes from  feces,  211 
method  of  obtaining  leucocytes  from, 

345 
Kabies,  900-909 

filtrability  of  virus,  905 
incubation  period  in,  900,  901 
Negri  bodies,  cultivation  of  by  No- 

guchi,  905,  906 
diagnosis  by,  902 
significance  of,  904,  905 
staining   of,   902-904 
specific  therapy,  906-909 
dosage  of,  908,  909 
preparation  of  the   cord,  907,  908 
" street"  virus,  901 
symptoms  in  animals,  901 
symptoms  in  man,  901 
"virus  fixe,"  901 

attenuation  of,  906 
Rage,  900.     See  also  Rabies 
Eat-bite  fever,  873 
Ratin,  692 

Rat  leprosy,  625,  526 
Rauschbrand,  768.    See  also   Symptom- 

matic  anthrax 
Reducing  powers  of  bacteria,  200 

method  of  testing  for,  200 
Refractive  index  of  bacteria,  26 
Relapsing  fever,  861 

cultivation     of      Obermeier's     spiro- 

chaete,  862 
immunity,  867 
morphology,  861 
pathogenicity,  864 
staining,  861 
transmission  of,  866 
types  of,  865 
Reproduction  of  bacteria,  19 

rate  of  growth,  19 
"  Resistance  "  defined,  240 

variations  in,  243 
Respiration  of  bacteria,  29 


Rheumatism,  gonorrheal,  553 

streptococcus  viridans  in,  422,  423 
Rhinoscleroma,  bacillus  of,  724 

pathogenicity  of,   725 
Rhizopoda,   1050 
Rhusiopathiae,  bacillus  of,  503 
Rickettsia  bodies,  957-960 
description  of  the  group,  958 
classification  of,  959 
R.  Prowazeki,  description  of,  960 
Dermacentroxenus  richettsi,  960 
staining  of,  958 
inheritance  of,  959 
in  typhus  fever,  942,  943,  944 
in  Rocky  Mountain  spotted  fever,  953 
in  trench  fever,  949 
Rideal-Walker  method  of  testing  disin- 
fectants, 98 
Ringworm  or  tinea, 

eczema  marginatum  and   pompholyx, 

1007 
epidermophyton  inguinale,  trichophy- 

ton  cruris,  1007 
microsporon,   1003 
cultures,  1004 
morphology,  1004,  1005 
other  members  of  this  group,  1005 
trichophyton,  1005 

classification,   1006 
Ringworm  group,  995-1000 
cultivation  of,  1000 

conservation  medium   (Sabouraud), 

1000 

test  medium  (Sabouraud),  1000 
cultural  characteristics,  996,  997 
immunity,  999 

methods  of  examination,  999 
morphology,  997 
pathogenicity,  998 
pleomorphism,  998 
Rocky  Mountain  spotted  fever,  950 
clinical   description,   950 
incubation  period  in,  950 
epidemiology,  951 

transmission  by  Dermacentor   ven- 

istus,  950 

transmission  to  guinea  pigs,  951 
transmission  to  rabbit,  951,  952 


1184 


INDEX  OF  SUBJECTS 


Kocky  Mountain  spotted  fever,  epidemi- 
ology, transmission  to  monkeys,  925 
Rickettsia  in,  952 
Boot  tubercles  in  the  leguminosae,  61 

SABOURAUD'S  medium  for  members  of 

the  ringworm  group,  1000 
Saccharomyces  albicans,  989 
Saccharomyces  hominis,  985 
Saccharomycetes  or  yeasts,  980 
Sachs-Georgi  reaction  for  syphilis,  328 
Salts  in  the  nutrition  of  bacteria,  31 
Saprophytes  defined,  33,  231 
Saprospira,  847 
Sarcina,  genus,  41 

in  the  normal  nose,  221 
Sarcodina  (Khizopoda),  1050 
Sarcophysematos  bovis,   768.     See  also 

Symptomatic  anthrax 
Sarcosporidia,  1135 
Scarlatina,   926-930.     See   also   Scarlet 

fever 
Scarlet  fever,  926-930 

diphtheroids  as  the  causative  agent, 

927 
streptococci   as   the   causative   agent, 

925,   927,   298 
epidemiology  of,  928 

transmission  by  nasal  and  pharyn- 

geal  mucous,  928 
period  of  infectiousness,  929 
"return"  cases,  929 
incubation  period,  929 
Schick  reaction,  577 
Schizomycetes,  definition  of,  41 
Schweineseuche,  829 
Sepsine,  52 

Septicaemia,  definition  of,  233 
Serum,  methods  of  obtaining  from  man, 

301,  205 

from  rabbits,  301 
from  large  animals,  302 
Serum  sickness,  366,  367 
Shiga's  bacillus  in  bacillary  dysentery, 

700,  704,  707 

Side  chain  theory,  Ehrlich,  216-268 
Sitotoxismus,  51 
Size  of  bacteria,  9 


Skin  reactions,  368 
delayed,  368 
for     determining     hypersensitiveness, 

369 

immediate,  368 

Sleeping  sickness,  African,  1086 
Sleeping     sickness.       See     Encephalitis 

lethargica 
Smallpox,  890-899 
etiology,  892-893 
relation  to  eowpox,  894 
vaccination,  898-899 
vaccine  production,  894 
calf  inoculation,  896 
collection   and   sterilization  of  the 

pulp,  897 

human-calf-rabbit  seed  virus,  896 
method  of  testing,  897-898 
' '  retro-vaccination, ' '  896 
''seed  virus,"  896 
Smegma  bacillus,  618 
identification  of,  619 
morphology  of,  618 
occurrence  of,  618 
Smith  fermentation  tube,  196 
Soil,  bacteria  in,  1012 

numerical  estimation  of,   1015 
pathogenic  bacteria  in,  1013 

persistence  of  after  the  burial  of 

infected  cadavers,  1014 
Specific  gravity  of  bacteria,  26 
Specimens  from  patients,  methods  of 

obtaining,  206,  207 
Spinal  fluid,  examination  of,  208-210 
in  acute  meningitis,  209,  528,  529 
in  poliomyelitis,  209 
in  syphilis,  209 
in  tuberculosis,  209 

Spiral  organisms,  classification  of,  847 
Spirillaceae,  definition  of,  42 
Spirillum,  description  of,  9 

genus,  42 
Spirillum    cholera)    asiaticae,    831.      See 

also  Cholera 
S.  Deneke,  845 
S.  Finkler-Prior,  845; 
S.  Massaua,  844 
S.  Metchnikovi,  844 


INDEX  OF  SUBJECTS 


1185 


Spirillum  Milleri  in  the  normal  mouth, 

219 

Spirochaeta  anserina,  872 
Spirochaeta  calligyrum,  873 
Spirochaeta  Duttoni,  866 
Spirochaeta  gallinarum,  871 
Spirochaete,  genus,  42 

anaerobic  tissues  tubes  for  cultivation 

of,  167 
Spirochaete  Icterohaemorrhagiae,  885-888 

causative  agent  of  Weil's  disease,  886 

cultivation  of,  886-887 
Spirochaete   macrodentium,   873 

microdentium,  873 
Spirochaete  of  Obermeier,  861 
Spirochaeta  pallida,  849-861 

animal  pathogenicity,  857 

cultivation  of,  856-857 

demonstration  of,  852-855 

Wassermann  reaction  with,  866 
Spirochaeta  pertenue,  870 
Spirochaeta  phagedenis,  872 
Spirochaeta  refringens,  846,  849 
Spirochaetes,  diseases 'caused  by,  846 

Kat-bite  fever,  873 

relapsing  fever,  861 

syphilis,  849 

Vincent's  angina,  867 

Yaws,  867 
Spironema,  848 

Vincenti  in  the  normal  mouth,  219 
Spontaneous  generation,  4,  5,  6 
Spores,  16 

conditions    necessary    for    formation, 
16 

destruction  of  by  boiling,  83 

destruction  of  by  fractional  steriliza- 
tion, 84 

destruction  of  by  steam  under  pres- 
sure, 85 

discovery  of,  6 

germination  of,  18 

loss  of  ability  to  form  spores,  37 

position  of,  17 

resistance  to  temperature  changes,  36 

not  related  to  arthrospores,  12,  16 

special  stain  for,  116 
Sporothroichosis,  992 


Spirosoma,  genus,  42 
Sporotrichum  (sporothrix),  993 

miuutissimum,  1009 
Spine,  991 

Sputum,  examination  of,  215 
collection  of,  215 
for  pneumococcus  typing,  215 
for  tuberculosis  examination,  215 
Sputum,  Petroff's  method  of  isolating 

tubercle  bacilli  from,  591 
methods  of  concentrating  tubercle  ba- 
cilli in,  589 
antiformin,  589 
Staining  of  bacteria,  113 
acid-fast,  124 

Baumgarten 's,  125 
Ehrlich's,  124 
Gabbet's,  124 
Hermann's,  126 
Pappenheim  ;s.   125 
Ziehl-Neilson,  124 
capsule  stains,  116 
Buerger,  117 
Hiss,  116 
Huntoon,  116 

Wadsworth's    for    smear   and    sec- 
tion,  118 
Welchi,  116 
differential    stains,    Gram's    method, 

121 

classification  by,   123 
discussion  of  chemical  basis  for, 

120 

Jensen's  modification,  122 
Paltauf's  modification,    121 
Sterling's  modification,  122 
discussion  of  chemical  principles  un- 
derlying,   114 
flagella  stains,  118 
van  Ermengen's,   119 
Loeffler's,  119 
Smith's    modification    of   Pitfield's 

method,  119 
in  tissues,   129 

Gram-Weigert,  130 

Loeffler's,  129 

Mallory's  eosin  methylene  blue,  131 

method  for  staining  acitomyces,  132 


1186 


INDEX  OF  SUBJECTS 


Staining  of  bacteria,  in  tissues,  Pappen- 

heimer's  method  for  staining  Gram 

positive  bacteria,  131 
polychrome  stains,  127 

Giemsa,  127 

Jenner,  127 

Wood's,  128 
Wright 's  modification  of  Leishman  's, 

127 
special  stains  for  polar  bodies,  126 

earbol  thionin,  126 

Neisser,  126 

toluidin  blue,   126 
spore  stains,  116 

Abbott's,  116 

Moeller's,   116 
stains  in  common  use,  115 

carbol-fuchsin,  115 

Loeffler's  alkaline  methylene  blue, 
115 

Pappenheim-Saathof  methyl  green, 
115 

toluidin  blue,  115 

Staphylococcus  epidermidis  albus,  397 
Staphylococcus  pyogenes  albus,  397 
Staphylococcus    pyogenes    aureus,    384- 

397 

agglutinins  in  immune  serum,  396 
cultural  characteristics,  385 
immunization  against,  396 

active,  396 

passive,  397 
in  the  mouth,  217 
in  the  nose,  221 
morphology,  384 
pathogenicity,  390 

in  animals,  390 

in  man,  391,  392 

impetigo  contagiosum,  392 

osteomyelitis,   392 

paronychia,  391 
pigment  formation,  388 
resistance  of,  388 
staining,  384 
thermal  death  point,  388 
toxic  products,  393 

hemolysins,  393 

thermolability  of,  394 


Staphylococcus  pyogenes   aureus,   toxic 

products,  leucocidin,  392 
thermolability  of,  395 
differentiation    from    leueotoxin, 

395 

virulence  of,  389 

Staphlycococcus  pyogenous  citreus,  397 
Sterigmocystis,  983 

Stomxys,  flies  in  poliomyelitis  transmis- 
sion, 918 
Strangles,  415 
Strauss  test  for  diagnosis  of  glanders, 

791 

Streak  plate,  technique  of,  178 
Streptothrix,   962.     See  also  Nocardia, 

964,  and  Actinomyces,  965 
genus,  42 

in  the  normal  mouth,  220 
of  Israeli,  965 
of  Eosenbach,  965 

Steam,  in  the  destruction  of  bacteria,  80 
live,  83 
saturated,  82 
"superheated,"'  82 
under  pressure,  85 
Stegomyia  fasciata  or  calopas,  877,  884 

habits  of,  883-884 
Sterling's  modification  of  Gram's  stain, 

122 

Sterilization,  fractional,  84 
practical  methods,  82 
' '  Arnold ' '  sterilizer,  83 
autoclave,  85 
boiling,  83 
burning,  82 
hot  air,  82 
Streptococcus  erysipelatis,  416 

genus,  41 

Streptococcus  hemolyticus   (Beta  type), 
408.     See  also   Streptococcus  pyo- 
genes below 
as  secondary   invaders   in   small   pox 

and  diphtheria 

grouping  by  agglutination,  412,  413 
hemolysin  production  by,  426 

thermolability  of,  427 
homogeneity  of  group  as  determined 
by  complement  fixation,  411,  412 


INDEX  OF  SUBJECTS 


1187 


Streptococcus   hemolyticus,   in   broncho- 
pneumonia  following  influenza,  419 

in  bronchopneumonia  following  mea- 
sles, 419 

in  epidemic  sore  throat,  421 

in  puerperal  sepsis,  417 

in  scarlet  fever,  419 

interference  of  with  suture  of  wounds, 
766 

septicaemia  caused  by,  418 

tonsilitis  caused  by,  418 

toxic  substances  produced  by,  425 
Streptococcus    mucosus-    (pneumococcus 

mucosus,  Type  III),  403,  see  also  448 

Streptococcus    pyogenes     (streptococcus 

hemolyticus  and  viridans),  401-445 

agglutinin  production  by,  433 

technique     of     agglutination    with 
spontaneously      agglutinating 
strains,  433,  434 

antibody  production,  429,  430 

antistreptococcic  sera,  430,  434 

standardization    of    by    protection 
experiments,  431 

capsule  formation  by,  403 

classification,  407 

by  fermentation  reactions,  409,  410 
Type  Alpha,  408 
Type  Beta,  408 
Type  Gamma,  408 

cultivation,  404,  405 

differentiation     from     pneumococcus, 
450 

in  the  mouth,  217 

in  the  nose,  221 

leucocidin  production  by,  429 

morphology,  402 

pathogenicity,   413 

nature  of  lesions  in  animals,  414 
nature  of  lesions  in  man,   415-423 

preservation  of,  407 

resistance  of,  406 
to  chemicals,  407 

staining,  402,  403 

thermal  death  point,  406 

variations  in  size,  403 

virulence  of,  413,  414 


Streptococcus  viridans  (Alpha  type), 
408,  409.  See  also  Streptococcus 
pyogenes 

heterogenity  of  group,  411,  412 
in  endocarditis,  422,  423 
relation  to  chorea,  423 
relation  to  rheumatic  fever,  422,  423 
relation  to  poliomyelitis,  423 
tissue  specificity  of,  424 
Strong  bacillus  in  bacillary  dysentary, 

704,  707 
Substances  of  unnkown  composition  in 

bacterial  nutrition,  32 
Sulphur  dioxide  as  a  fumigating  agent, 

106 

Sulphur  bacteria,  67 
Surra,  1080 

Symbiosis  of  bacteria,  35 
Symptomatic  anthrax,  bacillus  of,  758- 

'772 

cultivation,  770 
differentiation  from  vibrion  septique, 

759 

immunity,  771 
morphology,  769 
pathogenicity,  770 
staining,  769 
toxins,  771 
vaccines,  772 
Syphilis,  849-861 

spirochaeta  pallida  in,  850 
animal  pathogenicity,  857 
cultivation  of,  856,  857 
demonstration  of,  852,  855 
dark  field,  853 
in  smear,  853-855 
immunization  with,  859 
Wassermann  reaction  with,  866 


TARBARDILLO,  934,  937 

Temperature,   relation   of,   to   bacteria, 

36-39 

adaptation  to,  37 
effect  of  changes  in,  37 
effect   of   low   temperatures   on    bac- 
teria, 39 
resistance  of  spores,  38 


1188 


INDEX  OF  SUBJECTS 


Temperature,  relation  of,  to  bacteria, 
thermal  death  point  of  non-spore 
bearers,  38 

Terchloride  of  iodin,  93 
Tertian  fever,  1106 

clinical  description,  1115-1117 
Tetanus,  bacillus  of,  727-740 
antitoxin,  736-740 

immunization  of  horses,  274,  275 
prophylactic  use  of,  736 
standardization  of,  276 

L-J-  dose  of  toxin,  276 
therapeutic  use  of,  738 

administration  of,  740 
biological  considerations,  729 
distribution,  728 
morphology  of,  727 
pathogenicity  of,  734 
acute  form  of,  735. 
"  chronic "  form  of,  735 
incubation   period   after   infection, 

735 
symptoms  in  man,  735,  736 

atypical  cases,  726 
spores,  latency  of,  735 

resistance  of,  736 
staining  of,  727 
tetanolysin,  734 
toxin,  731 

action  of,  723 

incubation  period  with,  733 

L-j-  dose,  276 

medium  for  the  production  of,  731 

M.  L.  D.  for  guinea  pigs,  275 

M.  L.  D.  for  mice,  274,  732 

precipitation  of,  731,  732 

thermolability  of,  732 

destruction  of  by  eosin,  732 
types  of,  737 

relation  to  toxin  production,  738 
Tetramitis  mesnili,  1074 
Theileria  parva,  1131 
Thermoregulators,  193 
Thiothrix,  genus,  42 
Thread  reaction  of  Pfaundler,  283 
Throat,  smears  and  examinations,  216 
for  diphtheria,  216 
for  Vincent's  angina,  216 


Thrush,  989 
Timothy  bacillus,  617 
Tinea  or  ringworm,  1003-1008 
Tinea  vervicolor,  1009 
Tissue   in   culture   media,   reducing   ac- 
tion of,  189 

Tissues,  latency  of  bacteria  in,  222,  223 
Tissue  specificity,  discussion  of,  424 
Titration,  136 
colorimetric  method,  138 
actual  steps  in,  142 
''buffer/'  definition  of,  138 
indicators  for  various  ranges  of  Ph, 

141 

preparation  of  color  standards 
Ph,  meaning  of,  138,  139 
comparator,  for  reading,  142 
phenolphthalein,  old  method,  136 
Toluidin  blue  stain  for  polar  bodies,  115 
Tonsilitis,  streptococcus  in,  418 
Toxin  antitoxin  reaction,  nature  of,  255- 

268 

analogy  to  colloidal  reactions,  265 
Arrheinus  and  Madsen,  theories  con- 
cerning, 264,  265 
Ehrlich's  analysis  of,  257-263 
side  chain  theory,  266-268 
Toxin,  definition  of,  234 
Toxin,  diphtheria,  571-574 

antitoxin  reaction,  theoretical  consid- 
erations, 255-268 

chemical  and  physical  properties  of,  573 
Ehrlich's  analysis  of,  257-263 
deterioration  of,  258 
epetoxoid    or    toxin,    definition    of, 

261 

L+  dose,  260 
L0  dose,  259 
standardization,  258 
toxoid,  259 
unit  of,   257 

method  of  production,  571 
thermolability,  572 
Toxin,  tetanus,  731-733 
action  of,  733 
incubation  period  with,  733 
L+  doses,  276 
medium  for  the  production  of,  731 


INDEX  OF  SUBJECTS 


1189 


Toxin,    tetanus,    M.    L.   D.    for    guinea 

pigs,  274,  732 
M.  L.  D.  for  mice,  274 
precipitation  of,  731,  732 
thermolability  of,  732 

destruction  of  by  eosin,  732 
Trachoma,    influenza    bacillus    in,    500, 

501 
Transferring     cultures,     technique     of, 

173 

Trench  fever,  946-950 
clinical  description,  947 
transmission  and  etiology,  947-950 
incubation   period    of   louse   trans- 
mitted cases,  948 
louse  transmission,  948,  949 
Eickettsia  bodies  in,  949 
virus   contained  in   the   blood   and 

urine,  948 
Treponema,  848 
Treponema   macrodentium,    849 

occurrence  of  in  dirty  mouths,  219 
Treponema  pallidum,  851-861.    See  also 

Spirochaeta  pallida 
as   an   antigen   for   the   Wassermann 

test,  317,  860 
Trichomonas,  1073 
vaginalis,  1073 
intestinalis,  1073 
Trichomycetes,  definition  of,  961 

classification  of,  962 
Trichophyton,  1005 
Trichophyton  cruris,  1007 
Tricresol,  95 
Trillat  autoclave  for  the  generation  of 

formaldehyde,  107 
Triphenylmethane  dyes  as  disinfectants, 

97 

Trypanosoma  brucei,  1081 
cultivation  of,  1083 
morphology  of,  1081 
Nagana  caused  by,   1081 
transmission  of,  1082 
.Trypanosoma  cruzi,    1091 
Trypanosoma  equiperdum,  1084 

complement    fixation    for    diagnosis, 
1085 


Trypanosoma  evansi,  1080 

infection  in  horses,  mules,  etc.,  1080 
transmission  of,  1080 
Trypanosoma  gambiense  (sleeping  sick- 
ness), 1086 
clinical  signs,  1088 
etiology,  1088 

transmission  by  insects,  1088 
morphology,  1089 
pathogenicity,  1089 
Trypanosoma  hippicum,  1083 
morphology,  1083 
pathology,  1083 
Trypanosoma  lewisi,  1079 
insect  hosts  of,  1080 
morphology  of,  1079 
multiplication  in  rats,  1080 
Trypanosoma  rhodosiense,  1090 
diagnosis,  1090 
prophylaxis,  1091 
treatment,  1091 
Trypanosoma  rotatorium,  1078 
Trypanosoma  valenti,  1076 
Trypanosomidae,  1076 
Tubercle  bacillus  (avian),  614 
Tubercle   bacillus    (bovine    type),   612- 

614 
differentiation  of  from  human  type, 

514 
Tubercle   bacillus    (human   type),   586- 

612 
bacilli  closely  related  to,  612 

bacillus  of  bovine  tuberculosis,  612- 

614 

cultural  differentiation  from  hu- 
man type,  613  • 

differentiation  by  virulence,  613 
summary    of    differentiation    be- 
tween bovine  and  human  type, 
614 

bacillus  of  avian  tuberculosis,  614 
bacillus    of    tuberculosis    in    cold- 
blooded animals,  615 
immunization  with,  616 
bacillus,  timothy,  617 
bacillus    butyricus     (Butter    bacil- 
lus), 617 
biological  considerations,  593 


1190 


INDEX  OF  SUBJECTS 


Tubercle  bacillus   (human  type),  chem- 
ical analysis  of  tubercle  bacillus,  603 

complement  fixation  with,  608 

cultivation  of,  590-593 
Hesse's  medium  for,  593 

discovery  of,  586 

frequency  of,  597 

immunization,  passive,  611 
Maragliano's  serum,  612 
Marmorek's  serum,  612 

isolation  of,  590 
from  feces,  212 

Petroff' s  method  for,  from  sputum, 
591 

milk  in  the  transmission  of,  598 

morphology  of,  586 

pathogenicity,  593-597 

for  animals  (human  type),  597 
for    animals,    human    and    bovine 

type  compared,  613,  614 
lesions  produced  by,  595,  596 
manner  of  invasion,  594,  595 
occurrence  of  in  blood,  596 
tubercles,  description  of,  593 

prevention  of,  602,  603 

sputum,  method  of  concentrating  tu- 
bercle bacilli  in,  589 

staining,  587-589 

antiformin    for    concentrating    tu- 
bercle bacilli  in  sputum,  589 
Gabbett  's  method,  588,  124 
Herman's  method,  588,  126 
in  sections,  132 

Much's  method  for  Much's  gran- 
ules, 588 

Weiss'  modification  of,  589 
Pappenheim's  stain  for  differentia- 
tion from  smegma  bacillus,  589, 
125 

susceptibility   to   bovine   and   human 
types,  600 

thermal  death  point,  593 

toxins   produced   by,   604.     See   also 
Tuberculins 

transmission,  597 

by  butter,  1042,  1044 
by  dust,  601 
by  milk,  598 


Tubercle  bacillus   (human  type),  trans- 
mission, inheritance  of,  602 
predisposing  factors,  601 
tuberculins,  604 

bouillon  filtre  (Denys),  606 
diagnostic  use  of,  607 

cutaneous     tuberculin     reaction, 

608 

on  cattle,  609,  610 
ophthalmo-tuberculin      reactions, 

607 

subcutaneous,   607 
new,  605 
new    tuberculin-bacillary   emulsion, 

606 

old,  604 

reaction  to,  by  lepers,  625 
therapeutic  uses  of,  610 
Tubercle   bacillus    in   cold-blooded   ani- 
mals, 615 

immunization  with,  616 
Tuberculins,  diagnostic  use  of,  607 
for  testing  cattle,  1037 
preparation  of,  604-606 
therapeutic  use  of,  610 
Typhoid  bacillus  of,  643-685 
antibodies  produced  by,  659 
agglutinins,  662 

Widal  reaction,  663-667,  282,  302 
Dryer's  method,  666 
method  of  obtaining  patient 's 

serum,  665 

serum  dilutions  used,  663 
bactericidal  and  bacteriolytic,  661 
opsonins,  667 
precipitins,  667 
biological  considerations,  648 
blood  cultures  for,  214,  651 
carriers,  669-675 
chronic,  670-675 

gall-bladder  carriers,  671 
" healthy"  carriers,  673 
intestinal  carriers,  672 
liver  duct  carriers,  671 
urinary  carriers,  672 
pathological  lesions  caused  by  car- 
rier state,  674 


INDEX  OF  SUBJECTS 


1191 


Typhoid,  bacillus  of,  carriers,  stool  ex- 
aminations for  the  detection  of, 
653 

temporary  carriers,  670 
treatment  of,  675 
characteristics  of,  648 
cultivation  of,  643-648 

differential  media  for,  647,  158-165 
brilliant     green     agar      (Krum- 

wiede),  161 
brilliant       green       eosin       agar 

(Teague),  162 

Conradi-Drigalski  medium,  158 
Endo's  medium,  159 
discovery  of,  643,  8 
fermentations  of,  646,  648,  chart  of, 

718 
cultural  differences  within  typhoid 

group,  648 

immunity,  active,  659 
after  one  attack,  660 
method  of  immunizing  animals,  659 
vaccination,  680-684 
duration,  684 
lipovaccine,  683 
sensitized  typhoid  vaccine,  683 
isolation  of  from  feces,  653 
morphology  of,  643 
pathogenicity  of,  649-656 
for  animals,  649-650 

method  of  inducing  carrier  state 

in  animals,  650 
suppurative  lesions  due  to  typhoid 

bacillus,  655 

typhoid  fever  in  man,  650-656 
septicaemia  in,  650 

media    for     isolating    typhoid 

from  blood,  651 
typhoid  bacilli  in  the  rose  spots, 

655 

typhoid  bacilli  in  the  sputum,  655 
typhoid  bacilli  in  the  stool,  652 
typhoid    fever    without    intestinal 

lesions,  656 
poisons  of,  656,  659 

endotoxins,    method    of    obtaining, 

657 
prevention  of,  680 


Typhoid,  bacillus  of,  sanitary  considera- 
tions, 668-680 
transmission  of,  676 

by  contact -infection,  679 
by  flies,  679 
by  milk,  678 
by  oysters,  1047 
by  water,  677 

isolation  of  typhoid  from,  1023 
specific  treatment  of  typhoid  fever, 

684 

staining,  643 

thermal  death  point,  648 
Typhi  Murium,  bacillus  of,  691,  688 
Typhus  fever,  934-945 

clinical  description,  935,  936 

differential  diagnosis  from  purpura 

hemorrhagica,  935 
epidemiology,  936 

Serbian  epidemic,  938,  939 
transmission  by  lice,  937,  938 
etiology,  940 

Plotz  bacillus,   discussion   of,   941, 

942,  944 
probably    not    due    to    a    nltrable 

virus,  940 

Eickettsia  bodies  in,  942,  943,  944 
Rickettsia  prowazeki,  944 
immunity  in,  945 
incubation  period,  935 
prevention  of,  944 
transmission  to  guinea  pigs,  940 
transmission  to  monkeys,  939 
Weil-Felix   reaction   in   diagnosis   of, 

945 
Tryamin,  produced  in  bacterial  cultures, 

237 
Tyrotoxismus,  51 

UROBACILLUS  liquefaciens  septicus,  641 
Urine,  examination  of,  210 
Urticaria,  370 

U.  S.  Public  Health  Service,  method  of 
testing  disinfectants,  99 

VACCINATION,  small-pox,  898-899 
Vaccination,  typhoid  bacillus,  680-684 


1192 


INDEX  OF  SUBJECTS 


Vaccine  production  (small-pox),  894 

calf  inoculation,  896 

collection   and   sterilization   of   pulp, 
897 

humaii-calf-rabbit  seed  virus,  896 

method  of  testing,   897-898 

' l  retrovaccination, ' '  896 

"seed  virus/'  896 
Vaccine  therapy  (Wright),  342 

autogenous,  342 

dosage    in    staphylococcus    infection, 
344 

method  of  counting,  343 

negative  phase  in,  344 

positive  phase  in,  344 

production  of,  342 

Vaccinia,  or  cowpox,  relation  to  small- 
pox, 894 

Vanillin  test  for  indol,  199 
Variola  or  small  pox,  890-899.  See  Small- 
pox 
Varicella  or  chicken  pox  distinct  from 

smallpox,  894 

Venereal  diseases,  Public  Health  man- 
agement of,  556 
Vibrion  septique,  756 

antitoxin,  758 

discovery  of,  756 

differentiation  from  symptomatic  an- 
thrax, 759 

method  of  identifying,  753 

occurrence  of  in  war  wounds,  756 

pathogenicity  of,  738 

toxin  production,  757 

types  of,  757 
Vincent's  Angina,  867-869 

fusiform  bacilli,  in,  868 

in  throat  smears,  216 

spirilla  in,  868 
Vinegar,  making  of,  57 
Virulence, 

relation  to  capsulation,  14 

loss    of    due    to    cultivation    at    high 
temperatures,  39 

definition  of,  232 

problem  of,  348 

Bail's  aggressin  theory,  349 
arguments  against,  350 


Virulence,  problem  of,  Bail's  agressin 
theory,  relation  to  anaphylatoxin,  351 
* '  Virus    fixe ' '    in    rabies    therapy,    901 
Vitamines  in  bacterial  nutrition,  33 
Vulvovaginitis  due  to  gonococcus,  556 


WADSWOKTH   capsule   stain   for   smear* 

and  sections,  118 
Wassermaim  test  for  syphilis,  317 
actual  test,  322 
antigen,  317 

alcoholic   extract   of  heart   muscle, 
318 

cholestrinized,  318 

Noguchi's  acetone  insoluble  lipoid, 
318 

spirochaeta  pallida,  860 
complement  in,  321 
hemolytic  serum,  319-320 

production  of,  319 

titration  of,  320 

unit  of,  320 
modifications  of,  323 
serum  to  be  tested  for  syphilitic  anti- 
body, 321 

sheep  corpuscles,  321 
Water,  bacteria  in,  1016 

pathogenic  bacteria  in,  1016,  1017 

in  surface  water,  1017 

in  rain  and  snow,  1017 

in  "ground"  water,   1018,  1019 

quantitative  estimation  of,  1019,  1020 

standard  medium  for,  1020 

procedure,  1021 

expression  of  results,  1021 
qualitative  estimation,   1021 

typhoid  isolation  from,  1022 
Weil's  disease,  885-888 

clinical  description,  885-886 
etiology,       spirochseta      icterohsemor- 
rhagiae,   886 

cultivation  of,  886,  887 
prevention,  888 
serum  treatment,  888 
transmission,  887 
Weil-Felix  reaction,  non  specificity  of, 

942 


INDEX  OF  SUBJECTS 


1193 


Weil-Felix  reaction,  in  the  diagnosis  of 

typhus  fever,  945,  946 
Welch  bacillus,    751-756.     See  also   B. 

Welchii 

Welch's  capsule  stain,  116 
Welch  and  Nuttall,  method  of  rabbit  in- 
oculation for  the  isolation  of  anaer- 
obes, 211 
West    tube    in    taking    nasopharyngeal 

cultures,  542 
Whooping  cough,  bacillus  of,  504-508 

antibody  production  by,  508 

cultivation  of,  506 

epidemiology,  504,  505 

pathogenicity,  507 

morphology  of,  505 

staining  of,  506 
Widal  reaction,  282 

method  of  performing,  302 
Winckel's   disease,   633 
Wolbach's  table  of  disease   caused  by 

filtrable  viruses,  891 
Woolsorter's  disease,  782 
Wolfhugel  counting  plate,  195 
Wohlhynin  fever,  946.    See  also  Trench 

fever 

Wood's  polychrone  stain,  128 
Wood    ticks    in    the     transmission     of 

Rocky  Mountain  Spotted  fever,  952 
Wounds,  bacteriological  control  of,  763- 
768 

anaerobic  bacilli  found  in,  749-763 

cultural  examination,  766 

serological  treatment,  768 

smear  method,  Carrel,  754 

surgical  considerations,   767 
Wright's  method  of  counting  bacteria, 

195 


Wright's  stain,   127 

Wright 's  technique  of  the  opsonic  test, 

339 
vaccine  therapy,  341 

dose    in    staphylococcus    infections, 

344 

method  of  counting  bacteria,  343 
negative  phase,  344 
positive  phase,  344 

"X"    SUBSTANCES,    bacterial    poisons, 

237 
Xerosis  bacillus,  584 

YAWS,  870 

"Y"    bacillus   in    bacillary   dysentery, 

704,  707 
Yeasts,  980 

diseases  caused  by,  984,  985 

in  alcoholic  fermentation,  981 

in  the  normal  nose,  221 

other  yeast-like  parasites,  992 
Yellow  fever,  874-884 

etiology,  879-883 
early  claims,  880 
Noguchi's  work,  881-883 

distribution  of,  874 

immunity  in,  884 

prevention  of,  883 

transmission  of,  874-878 

ZIEHL-NEILSON  stain  for  the  tubercle 
bacillus,  124 

Zur  Nedden's  bacillus,  509 

Zymase,  enzyme  of  alcoholic  fermenta- 
tion, 58,  981 

Zymonema  Gilchristi,  985 


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